Chemiluminescent polymer with high selectivity on singlet oxygen as well as preparation method and application of chemiluminescent polymer

Abstract

The invention discloses a chemiluminescent polymer with high selectivity on singlet oxygen as well as a preparation method and application of the chemiluminescent polymer. The chemiluminescent polymer is a functionalized polyacrylamide high-molecular compound generated by carrying out free radical polymerization reaction on acrylamide, 5-acrylamido fluorescein and 4-acrylamido phthalylhydrazine, and has excellent water solubility, and the solubility is greater than or equal to 2.5 g / L. When the polymer is oxidized by singlet oxygen in a dark place, macroscopic yellow-green strong chemiluminescence is generated, and the emission wavelength peak value is 531 nm; the compound has extremely high chemiluminescence selectivity on singlet oxygen under physiological conditions, and singlet oxygen can be recognized at a submicromole level. Therefore, the polymer provided by the invention can be used as a novel polymer chemiluminescence probe for high-selectivity and high-sensitivity identification and detection of singlet oxygen under physiological conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of chemiluminescence detection, and specifically relates to a functionalized polyacrylamide chemiluminescent polymer, a preparation method of the polymer and an application of the polymer in selectively identifying singlet oxygen. Background Art

[0002] Singlet oxygen ( 1 O2) is an excited oxygen molecule with extremely high reactivity that is continuously produced during biological metabolism. This highly reactive transient intermediate has an important impact on human health. It can penetrate the mitochondrial membrane or nuclear membrane and react rapidly with many biological molecules such as DNA, lipids, free amino acids and proteins. It is the main bactericidal agent of the immune system of organisms and can be used in photodynamic therapy for cancer.

[0003] Under normal physiological conditions, cells 1 The production and removal of O2 are in a dynamic equilibrium state and maintain a very low concentration level, which will not harm the body. However, when the balance of production and removal in cells is disrupted, high concentrations of O2 1 O2 will have a strong toxic effect on organisms, causing membrane lipid peroxidation or delipidation, DNA damage, signal transduction disorders, affecting normal cell division, and in severe cases, causing cell death and other harmful effects, thereby causing various diseases such as cancer, body inflammation, tissue peroxidation, tumors and chemical poisoning. Therefore, 1 The detection and characterization of O2 is of great significance.

[0004] Existing tests 1 O2 detection methods mainly rely on various spectral techniques, such as electron paramagnetic resonance spectroscopy, phosphorescence, spectrophotometry, fluorescence spectroscopy and chemiluminescence, among which fluorescence and chemiluminescence are the two most widely used spectral detection methods. Chemiluminescence overcomes the background and scattering problems that cannot be avoided in fluorescence and has a higher signal-to-noise ratio. Therefore, it is very suitable for the detection of substances with extremely short lifespan and extremely low content in cells and living organisms. 1 O2 detection has more advantages. Development 1 O2 has a highly sensitive and selective chemiluminescent probe, especially under physiological conditions. 1 O2 chemiluminescent probes with high sensitivity and selective response are of great value to related life and medical research, life analysis and clinical diagnosis. Summary of the invention

[0005] The purpose of the present invention is to provide a chemiluminescent polymer with high selectivity and high sensitivity to singlet oxygen.

[0006] The structural formula of the chemiluminescent polymer provided by the present invention is as follows:

[0007]

[0008] In the formula, the molar ratio of x:y:z is 2.5-10:100:10; and the viscosity average molecular weight of the chemiluminescent polymer is 49000-55000.

[0009] The preparation method of the above chemiluminescent polymer consists of the following steps:

[0010] Step 1: Using N,N-dimethylformamide (DMF) as solvent and sodium bicarbonate (NaHCO3) as acid binding agent, isoluminol and acryloyl chloride are reacted to obtain 4-acrylamidophthaloyl hydrazide; the synthetic route is as follows:

[0011]

[0012] Step 2: Use acrylamide, 5-acrylamidofluorescein and 4-acrylamidophthaloyl hydrazide as monomers, DMF as solvent, 2,2'-azobisisobutyronitrile (AIBN) as initiator, and carry out polymerization reaction under the protection of inert gas. After the reaction, separate the orange-yellow solid to obtain a chemiluminescent polymer; the synthesis route is as follows:

[0013]

[0014]

[0015] In the above step 1, the molar ratio of isoluminol to acryloyl chloride and sodium bicarbonate is preferably 1:1.1-1.3:1.2-1.8.

[0016] Furthermore, in the above step 1, the temperature for the reaction of isoluminol and acryloyl chloride is 0 to 5° C., and the reaction time is 40 to 80 min.

[0017] In the above step 2, the molar ratio of acrylamide to 4-acrylamidophthalhydrazide and 5-acrylamidofluorescein is preferably 100:10:2.5-10, wherein 5-acrylamidofluorescein is synthesized according to the method in the document “Analyst, 2023, 148, 5942–5948.”

[0018] In the above step 2, preferably, the amount of 2,2'-azobisisobutyronitrile added is 0.8% to 1.2% of the molar amount of acrylamide.

[0019] In the above step 2, the inert gas is argon or nitrogen.

[0020] Furthermore, in the above step 2, the polymerization reaction temperature is 55 to 65° C., and the polymerization reaction time is 30 to 60 min.

[0021] The present invention also provides the use of the chemiluminescent polymer in selectively identifying singlet oxygen. The chemiluminescent polymer is 1 When oxidized by O2, it can produce yellow-green intense chemiluminescence visible to the naked eye, with a peak emission wavelength of 526nm; and under physiological conditions, 1 O2 has extremely high chemiluminescence selectivity and can be identified at the sub-micromolar level 1 O2.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1. The chemiluminescent polymer of the present invention is synthesized in one step by free radical polymerization reaction. The polymerization method is reliable and easy to operate, and the composition of the polymer can be flexibly adjusted by the amount of monomers used.

[0024] 2. The chemiluminescent polymer of the present invention grafts the cyclic hydrazide structure (isoluminol) as the chemiluminescent energy donor and the luminescent energy acceptor molecule (fluorescein) with high fluorescence quantum yield onto the side chain of the acrylamide polymer molecule through covalent bonds, and the cyclic hydrazide structure (isoluminol) as the chemiluminescent energy donor and the luminescent energy acceptor molecule (fluorescein) with high fluorescence quantum yield are spatially adjacent to each other, thereby fully ensuring the energy transfer efficiency between the energy donor and the energy acceptor, obtaining a highly amplified chemiluminescent signal, and there is no need to worry about the leakage of the chemiluminescent reagent during use.

[0025] 3. The chemiluminescent polymer of the present invention is 1 When oxidized by O2, it can emit strong chemiluminescence visible to the naked eye, and it is 1 O2 is extremely selective and can respond at sub-micromolar levels 1 O2 has the function of characterizing the physiological system 1 Unique properties of O2. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the infrared spectrum of the chemiluminescent polymer prepared in Example 1.

[0027] Figure 2 The chemiluminescent spectrum of the chemiluminescent polymer prepared in Example 1 and the chemiluminescent spectrum of the polymer 1 Chemiluminescent photograph of O2 oxidation (inset).

[0028] Figure 3 The chemiluminescent polymer prepared in Example 1 is 1 Selectivity for O2.

[0029] Figure 4 The chemiluminescence of the chemiluminescent polymer prepared in Example 1 varies with the pH of the medium.

[0030] Figure 5The chemiluminescent polymer prepared in Example 1 is 1 O2 response. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0032] Example 1

[0033] Step 1: Add 3.54g (20.0mmol) of isoluminol to a dry 50mL single-necked flask, add 10mL of DMF to dissolve it, and then add 2.52g (30.0mmol, 1.5eq) of sodium bicarbonate. After it is dissolved, stir for 30min under ice bath conditions, then add 2.05mL (24.2mmol, 1.2eq) of acryloyl chloride, remove the ice bath, stir at 0-5°C for 1h, pour the resulting reaction solution into ice water, add hydrochloric acid to neutralize the reaction solution, filter, and dry the filter cake in a vacuum oven at 50°C to constant weight to obtain 2.09g of 4-acrylamidophthaloyl hydrazide with a yield of 44.8%. This product is directly used in the next step of synthesis without separation.

[0034] Step 2: 0.231 g (1.0 mmol, 10.0 eq) of 4-acrylamidophthalhydrazide, 0.202 g (0.5 mmol, 5.0 eq) of 5-acrylamidofluorescein, 0.71 g (10 mmol, 100.0 eq) of acrylamide and 5 mL of DMF were added to a dry 50 mL three-necked flask. After stirring at room temperature to dissolve, 0.016 g (0.1 mmol, 1.0 eq) of 2,2'-azobisisobutyronitrile dissolved in 1 mL of DMF was added. Under argon protection, the oil bath was heated to 60°C for 45 min. The reaction mixture was then poured into 20 mL of methanol and filtered. The filter cake was placed in a vacuum oven at 50°C and dried to constant weight to obtain 0.632 g of an orange-yellow solid, i.e., a chemiluminescent polymer, with a yield of 55.3%.

[0035] Depend on Figure 1 In the infrared spectrum, 3325.69cm -1 The stretching vibration of hydroxyl group is 3183.21cm -1 The symmetric and asymmetric stretching vibrations of amide NH are represented at 2932.42 cm -1 The symmetric and asymmetric stretching vibrations of methylene and methine are at 1647.17358 cm -1 represents C=O symmetric stretching vibration, 1603.00cm -1 represents the vibration of the aromatic ring skeleton, 1446.26cm -1 The bending vibration of the methylene group near the carbonyl group is 1407.79 cm-1 The CH plane bending vibration is at 1313.75, 1181.23, 1102.87cm -1 The stretching vibration of CO bond is 484.46cm -1 represents COOH, indicating that the chemiluminescent polymer was successfully synthesized.

[0036] According to the national standard GB12005.1-89, at 30±0.05°C, in 1.0 mol / L NaCl aqueous solution, the intrinsic viscosity [η] of the obtained chemiluminescent polymer was measured by the one-point method. The formula provided by the national standard GB / T12005.10-92 is: Its viscosity average molecular weight is calculated to be 49749. After calculation, in the following structural formula, x=22, y=440, and z=44.

[0037]

[0038] Example 2

[0039] In step 2 of this example, the amount of 5-acrylamide fluorescein used is 0.101 g (0.25 mmol, 2.5 eq), and the other steps are the same as those in example 1 to obtain a chemiluminescent polymer. After testing, the viscosity average molecular weight of the chemiluminescent polymer is 54322, and after calculation, x=13, y=520, and z=52 in the structural formula.

[0040] Example 3

[0041] In step 2 of this example, the amount of 5-acrylamide fluorescein used is 0.303 g (0.75 mmol, 7.5 eq), and the other steps are the same as those in example 1 to obtain a chemiluminescent polymer. After testing, the viscosity average molecular weight of the chemiluminescent polymer is 49749, and after calculation, x=30, y=400, and z=40 in the structural formula.

[0042] Example 4

[0043] In step 2 of this example, the amount of 5-acrylamide fluorescein used is 0.404 g (1.0 mmol, 10.0 eq), and the other steps are the same as those in example 1 to obtain a chemiluminescent polymer. After testing, the viscosity average molecular weight of the chemiluminescent polymer is 54322, and after calculation, x=40, y=400, and z=40 in the structural formula.

[0044] Example 5

[0045] The chemiluminescent polymer prepared in Example 1 selectively recognizes 1 Applications in O2

[0046] Fix the coil on the light window of the fluorescence spectrophotometer, shield the excitation light with a black baffle, and place the 6.0×10 -4 g / mL chemiluminescent polymer solution (prepared with 0.01 mol / L NaOH aqueous solution) and 2.0×10 -3 mol / L NaClO solution, 2.0×10 -3 mol / L H2O2 solution (both NaClO solution and H2O2 solution were prepared with 0.01mol / L PBS buffer solution with pH=7.4) was pumped into the reaction coil by a peristaltic pump and mixed at the three-way connection. The chemiluminescence spectrum was scanned in the range of 300-600nm (incident slit: closed; exit slit: 20nm; PMT: -500V). Figure 2 The results of the measurement showed that the chemiluminescent polymer of Example 1 was 1 The chemiluminescence wavelength produced by O2 oxidation is in the range of 520-550nm, and the maximum emission wavelength is 531nm.

[0047] Under light-shielding conditions, 2.0 mL of 6.0 × 10 -4 g / mL chemiluminescent polymer solution into a vial and add 2.0 mL of 2.0×10 -3 mol / L NaClO solution and 2.0mL 2.0×10 -3 mol / L H2O2 solution, and use the continuous shooting mode of the digital camera to obtain multiple photos. Figure 2 The inserted picture is the one with the strongest luminescence, which shows that the chemiluminescent polymer of Example 1 is 1 When oxidized by O2, it produces yellow-green chemiluminescence visible to the naked eye.

[0048] The intermittent flow injection method was used to record the chemiluminescence intensity of 5 common active oxygen species reacting with the chemiluminescent polymer of Example 1 using a fluorescence spectrophotometer. 1 O2 concentration: 1.0×10 -3 mol / L, from 2.0×10 -3 mol / L NaClO solution and 2.0×10 -3 mol / L H2O2 solution was mixed with equal volumes to generate the product online and immediately react with 6.0×10 -4 g / mL chemiluminescent polymer solution mixed reaction; ClO - Concentration: 1.0×10 -3 mol / L, prepared with 0.01mol / L PBS buffer, pH=7.4; H2O2 concentration: 1.0×10 -3 mol / L, prepared with 0.01mol / L PBS buffer at pH=7.4; ·OH: 1.0×10-3 mol / LFeSO4 solution and 1.0×10 -3 mol / L H2O2 solution (both solutions were prepared with 0.01 mol / L PBS buffer, pH = 7.4) was mixed in equal volumes and reacted online and immediately with 6.0×10 -4 g / mL chemiluminescent polymer solution mixed reaction; By 1.0×10 -3 mol / L purine solution and 0.5 μg / mL xanthine oxidase solution (both solutions were prepared with 0.01 mol / L PBS buffer, pH = 7.4) were mixed in equal volumes and oxidized for 1 min, then mixed with 6.0×10 -4 g / mL chemiluminescent polymer solution mixed reaction. In the above chemiluminescent reaction, the volume of solution pumped into each pipeline is 2.0mL, and the volume of chemiluminescent polymer solution + active oxygen solution is equal to 6.0mL. Figure 3 The results showed that under the above conditions, the chemiluminescent polymer of Example 1 was 1 The chemiluminescence intensity produced by O2 oxidation is - 116 times of oxidation, H2O2, ·OH, No chemiluminescent signal was detected during oxidation, indicating that the chemiluminescent polymer of Example 1 was 1 O2 is highly selective.

[0049] The chemiluminescent polymer of Example 1 was recorded by a chemiluminescent analyzer in different pH buffer media (BR buffer solution adjusted to pH 2-12). 1 The chemiluminescent intensity of O2 oxidation was measured at a chemiluminescent polymer concentration of 6.0×10 -5 g / mL, 1 O2 concentration: 1.0×10 -4 mol / L, PMT = -500V. Figure 4 The results showed that the chemiluminescent polymer of Example 1 was 1 The chemiluminescence intensity generated when O2 is oxidized is the highest, indicating that the chemiluminescent polymer of Example 1 can be used as a polymer chemiluminescent probe for highly selective and sensitive identification and detection of singlet oxygen under physiological conditions.

[0050] In a PBS buffer solution at pH = 7.4, 2.0 mL of the chemiluminescent polymer solution of Example 1 was taken for each measurement. The final concentration of the chemiluminescent polymer was 6.0 × 10 -5 g / mL; H2O2 concentration: 1.0×10 -3 mol / L; 1 O2 concentration is determined by the added ClO -The concentration was determined from 1.0 μmol / mL to 10 μmol / mL. Figure 5 As shown in Figure 2, the detection limit calculated according to IUPAC is 0.28 μmol / mL, indicating that the chemiluminescent polymer of Example 1 is effective for 1 The detection of O2 has extremely high sensitivity.

Claims

1. A chemiluminescent polymer having high selectivity for singlet oxygen, characterized in that: The structural formula of the chemiluminescent polymer is shown below: In the formula, the molar ratio of x:y:z is 2.5-10:100:10; and the viscosity average molecular weight of the chemiluminescent polymer is 49000-55000.

2. A method for preparing the chemiluminescent polymer according to claim 1, characterized in that: The preparation method consists of the following steps: Step 1: Using N,N-dimethylformamide as solvent and sodium bicarbonate as acid binding agent, isoluminol and acryloyl chloride are reacted to obtain 4-acrylamidophthaloyl hydrazide; Step 2: using acrylamide, 5-acrylamidofluorescein and 4-acrylamidophthaloyl hydrazide as monomers, N,N-dimethylformamide as solvent, and 2,2'-azobisisobutyronitrile as initiator, a polymerization reaction is carried out under the protection of an inert gas, and an orange-yellow solid is separated after the reaction to obtain a chemiluminescent polymer; 3. The method for preparing a chemiluminescent polymer according to claim 2, characterized in that: In step 1, the molar ratio of isoluminol to acryloyl chloride and sodium bicarbonate is 1:1.1-1.3:1.2-1.

8.

4. The method for preparing a chemiluminescent polymer according to claim 2, characterized in that: In step 1, the temperature for the reaction of isoluminol and acryloyl chloride is 0-5° C., and the reaction time is 40-80 min.

5. The method for preparing a chemiluminescent polymer according to claim 2, characterized in that: In step 2, the molar ratio of acrylamide to 4-acrylamidophthalhydrazide and 5-acrylamidofluorescein is 100:10:2.5-10.

6. The method for preparing a chemiluminescent polymer according to claim 2, characterized in that: The added amount of the 2,2'-azobisisobutyronitrile is 0.8% to 1.2% of the amount of the acrylamide substance.

7. The method for preparing a chemiluminescent polymer according to claim 2, characterized in that: In step 2, the inert gas is argon or nitrogen.

8. The method for preparing a chemiluminescent polymer according to claim 2, characterized in that: In step 2, the polymerization reaction temperature is 55-65° C., and the polymerization reaction time is 30-60 min.

9. Use of the chemiluminescent polymer according to claim 1 in the selective recognition of singlet oxygen for non-diagnostic and therapeutic purposes.

Images