CN110128566B - Near-infrared fluorescent polymer probe for identifying formaldehyde and preparation method and application thereof - Google Patents
Near-infrared fluorescent polymer probe for identifying formaldehyde and preparation method and application thereof Download PDFInfo
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- CN110128566B CN110128566B CN201910363791.7A CN201910363791A CN110128566B CN 110128566 B CN110128566 B CN 110128566B CN 201910363791 A CN201910363791 A CN 201910363791A CN 110128566 B CN110128566 B CN 110128566B
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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Abstract
The invention discloses a near-infrared fluorescent polymer probe for identifying formaldehyde and a preparation method and application thereof, and particularly discloses a near-infrared fluorescent polymer probe with a structure shown in a formula I. The polymer probe provided by the invention is formed by carrying out chemical reaction on near-infrared fluorescent molecules and biocompatible chitosan, wherein the near-infrared fluorescent molecules are used as near-infrared fluorescent response units and can carry out chemical reaction with formaldehyde to emit red light so as to realize specific response to the formaldehyde; the chitosan is used as a main chain constitutional unit of the polymer, so that the polymer probe has good water solubility and biocompatibility. The near-infrared fluorescent polymer probe provided by the invention can be used for formaldehyde detection and imaging in aqueous solution, living cells and living bodies, and has important application prospects in the aspects of environmental monitoring, biological imaging and sensing.
Description
Technical Field
The invention belongs to the field of high molecular materials and chemical sensors, and particularly relates to a near-infrared fluorescent polymer probe for identifying formaldehyde and a preparation method and application thereof.
Background
Formaldehyde is a colorless gas with a special pungent odor, is recognized to have strong carcinogenic and carcinogenic effects, and is harmful to the eyes, nose, respiratory tract, skin and other organs that are easily exposed. At present, formaldehyde in a living environment mainly comes from human production activities such as phenolic aldehyde and urea-formaldehyde resin, textile auxiliaries, preservatives and the like, and metabolism and degradation of certain organic compounds. Formaldehyde is also an endogenous substance in the human body and can be generated through catalysis of biological enzymes such as semicarbazide-sensitive amine oxidase and lysine-specific histone demethylase 1. Research finds that formaldehyde plays an important role in the formation of spatial memory and cognitive abilities of humans. However, when the concentration of formaldehyde in blood exceeds the standard, some diseases such as cancer, nerve tissue degeneration, diabetes, senile dementia, chronic liver dysfunction, etc. may occur. Therefore, it is of great practical significance to effectively monitor or monitor formaldehyde in biological or environmental samples.
At present, some small-molecule fluorescent probes for formaldehyde detection have been reported. The maximum fluorescence emission wavelength of the probes involved in patent CN201610077333 and patent CN2015109227960 is only 530nm, and is far from the ideal near infrared imaging window 650-900nm, which brings phototoxicity to cells and also induces tissue autofluorescence interference (the emission light with short wavelength becomes the excitation light of the biological fluorescent molecules in the tissue); most of reported fluorescent probes are non-water-soluble fluorescent organic small molecules, and the fluorescent small molecules are easily damaged by biological enzymes in biological environments, so that the application of the fluorescent small molecules in the fields of life sciences, medicine and the like is limited. Therefore, the design and synthesis of the probe are suitable for detecting formaldehyde in a solution environment and detecting the level of formaldehyde in cells, the probe is small in using amount, safe and low in toxicity, and monitoring of formaldehyde in cells is realized so as to further assist in research on treatment and pathology of corresponding diseases.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a near-infrared fluorescent polymer probe for identifying formaldehyde and a preparation method and application thereof. The core of the invention is to utilize the near-infrared fluorescence of squaraine and the biocompatibility of chitosan. Compared with a small-molecule fluorescent probe, the polymer probe provided by the invention is based on a chitosan polymer chain which is cheap and easy to obtain, and is used for enriching low-concentration formaldehyde pollutants by utilizing the synergistic combination of a plurality of hydrazine-squaraine recognition sites and adjacent hydroxyl groups. Therefore, the specific chemical reaction of the formaldehyde and the probe is remarkably accelerated, the fluorescence response time is shortened, and the response sensitivity is improved.
The invention provides a near-infrared fluorescent polymer probe for identifying formaldehyde, which solves the problems of cytotoxicity, stability and water solubility of the existing formaldehyde sensor. The near-infrared fluorescent polymer probe for identifying formaldehyde has a general formula structure as shown in formula I:
formula I.
The second purpose of the invention is to provide a preparation method of the near-infrared fluorescent polymer probe for identifying formaldehyde, which comprises the following specific steps: chitosan is used as a polymer main chain, and a squaraine derivative for identifying formaldehyde, namely an infrared fluorescent molecule, is introduced through amidation reaction to obtain the chitosan derivative containing hydrazine-squaraine derivative groups.
Preferably, the near-infrared fluorescent molecule has a structure of formula ii:
and (5) formula II.
Preferably, the chitosan and the near-infrared fluorescent molecule II are in parts by weight: 1-3 parts of chitosan and 5-10 parts of near-infrared fluorescent molecules II.
Preferably, the molecular weight Mw of the chitosan is 5-20 kDa, and the deacetylation degree is 0.8-0.95.
Preferably, the combined catalyst is N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide.
Preferably, the molar ratio of the N-hydroxysuccinimide, the near-infrared fluorescent molecule II and the 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 1:1: 3-5.
Preferably, the acylation reaction is carried out at room temperature for 12-48 h.
The third purpose of the invention is to provide an application of the near-infrared fluorescent polymer probe for identifying formaldehyde in the specific detection of formaldehyde in the aqueous solution.
The fourth purpose of the invention is to provide an application of the near-infrared fluorescent polymer probe for identifying formaldehyde in the specific detection of endogenous or exogenous formaldehyde of cells.
Compared with the prior art, the invention has the advantages that:
1) the near-infrared fluorescent polymer probe for identifying formaldehyde is biocompatible, and the chitosan component in the probe structure ensures the water solubility and the cell compatibility of the probe;
2) the chromophore of the near-infrared fluorescent polymer probe for identifying formaldehyde is combined in a macromolecule by a chemical bond and is not easy to fall off;
3) the near-infrared fluorescent polymer probe for identifying formaldehyde provided by the invention has the advantages of uniform chromophore distribution, stable content, good luminescence performance and good photoconductive performance;
4) the near-infrared fluorescent polymer probe for identifying formaldehyde provided by the invention has good light stability, the absorption wavelength is 663nm, the emission wavelength is 698nm, and the near-infrared fluorescent polymer probe has fluorescent off-on response within the range of 650 plus 900nm of a biological safety near-infrared (NIR) window, so that the interference of a detection background on a result during detection can be greatly eliminated, and the detection accuracy is improved;
5) the 'off-on' type formaldehyde probe of the squaraine dye provided by the invention has good response to formaldehyde solution, can realize the detection of formaldehyde in cells, and has the advantages of low cost, sensitive response, easiness in popularization and application and the like.
Drawings
FIG. 1 is a UV-VIS absorption spectrum of a near-IR fluorescent polymer probe for identifying formaldehyde according to the present invention;
FIG. 2 is a graph of the change of the fluorescence spectrum of the probe according to the addition of different equivalent amounts of formaldehyde in example 1 of the present invention, wherein the fluorescence spectra are from bottom to top with formaldehyde concentrations of 0, 2, 4, 6, 8, 10, 12, 16, and 32. mu. mol/L;
FIG. 3 is a spectrum of the change of fluorescence intensity at 698nm with time within 1min of the probe and 2 equivalents of formaldehyde in example 1 of the present invention;
FIG. 4 is a bar graph of fluorescence data showing the selectivity of probes for different interfering analytes in example 1 of the present invention; in the figure, 1 cysteine, 2 glutathione, 3 acetaldehyde, 4 glyoxal, 5 benzaldehyde, 6 formaldehyde and 7 glucose;
FIG. 5 is a graph of fluorescence imaging of formaldehyde response in HeLa cells of the probe of example 1, wherein FIG. 5(a) is a reference group and FIG. 5(b) is an experimental group.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
The molecular structural formula of the near-infrared fluorescent polymer probe for identifying formaldehyde is as shown in formula I:
formula I
Wherein n =30 ~ 120.
The preparation method of the near-infrared fluorescent polymer probe for identifying formaldehyde provided by the invention comprises the following steps:
the method comprises the steps of taking chitosan as a polymer main chain, taking near-infrared fluorescent molecules as activatable fluorescent functional molecules, and introducing the molecules with the functions of specifically identifying formaldehyde and turning on the near-infrared fluorescent functional molecules into the chitosan through amidation reaction between amino groups of the chitosan and carboxyl groups of the near-infrared fluorescent molecules to obtain the near-infrared fluorescent polymer probe capable of identifying formaldehyde.
The near-infrared fluorescent molecule used in the invention has a structure of a formula II:
formula II
The chitosan is natural biological macromolecule, has wide source, safety, no toxicity and low price, and has good biocompatibility and biodegradability compared with synthetic macromolecule, so the prepared polymer probe as a formaldehyde probe in solution or cells has high environmental friendliness and biological safety by taking the chitosan as a raw material.
Example 1 preparation of near-infrared fluorescent Polymer Probe for Formaldehyde recognition
3.0g of chitosan (Mw =10kDa, DA = 0.85) was dissolved in 25mL N, N' -Dimethylformamide (DMF), 5.0g of near-infrared fluorescent molecule ii and 5mL of N-hydroxysuccinimide (NHS) containing 0.01mol were added under nitrogen-passing protection, and after stirring, 0.04mol of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) was added, and the reaction was stirred at room temperature for 24 hours. Dialyzing in deionized water for two days, vacuum freeze drying to obtain target probe, testing in ultraviolet absorption spectrometer to obtain ultraviolet-visible absorption spectrum shown in figure 1, with the maximum absorption peak of visible target near infrared fluorescent probe in water solution being 663 nm.
Example 2 fluorescence spectra changes of near-infrared fluorescent polymer probes reacted with different equivalents of formaldehyde
The probe prepared in example 1 was dissolved in DMF to prepare a probe mother liquor with a concentration of 0.5mmol/L (the concentration of the probe was 0.5 mmol/L); adding the formaldehyde solution with the mass fraction of 37% into distilled water to prepare formaldehyde mother liquor with the formaldehyde concentration of 1 mmol/L. 60 μ L of the obtained solution was taken out of the probe mother liquor and added into a 5mL centrifuge tube, and different equivalents (0-2 eq) of formaldehyde mother liquor, which is a multiple of the number of moles of formaldehyde in the formaldehyde mother liquor relative to the number of moles of the probe in the probe mother liquor, were added, and 1.44mL of DMF and different volumes of PBS aqueous solution with a concentration of 25mmol/L, pH 7.4.4 were diluted to 3mL to prepare a test solution with a probe concentration of 10 μmol/L and containing 50% DMF. The fluorescence spectrometer was used to measure the change of fluorescence spectra (excitation wavelength 650 nm) between the probe and formaldehyde reaction solutions of different equivalent weights, as shown in FIG. 2. As can be seen from FIG. 2, the fluorescence peak at 698nm of the near-infrared fluorescent polymer probe solution of the present invention is gradually increased with the gradual increase of the formaldehyde addition equivalent. When the fluorescence intensity reaches the maximum value, the fluorescence intensity is enhanced by 7.4 times compared with that of the probe blank liquid.
The ultraviolet-visible absorption and fluorescence emission tests show that the absorption and emission wavelengths of the near-infrared fluorescent probe are in the near-infrared region.
Example 3 fluorescence Change with Formaldehyde at different times for near Infrared fluorescent Polymer probes
60. mu.L of the fluorescent probe stock solution obtained in example 2 was taken out and put into a 5mL centrifuge tube, and 60. mu.L of a formaldehyde stock solution having a concentration of 0.5mmol/L was added thereto, followed by stirring and dilution to 3mL with 1.44mL of an aqueous PBS solution (25 mmol/L concentration, pH 7.4) to prepare a test solution having a probe concentration of 10. mu. mol/L, a formaldehyde concentration of 3mmol/L and a DMF content of 50%. The fluorescence spectrum was measured with time using an excitation wavelength of 650nm, and the results are shown in FIG. 3. As can be seen from FIG. 3, the fluorescence intensity at 698nm gradually became larger with time and reached a maximum around 30 seconds.
Example 4 Selective investigation of near Infrared fluorescent Polymer probes for different interfering analytes
60 μ L of the fluorescent probe stock solution from example 2 was added to a 5mL centrifuge tube and the following different concentrations of analyte were added: 20 μmol/L formaldehyde, 0.5mmol/L acetaldehyde, 50 μmol/L glyoxal, 1mmol/L benzaldehyde, cysteine, glutathione, glucose, diluted to 3mL with 1.44mL DMF and various volumes of PBS aqueous solution (concentration 25mmol/L, pH 7.4) were prepared as a test solution with a probe concentration of 10 μmol/L containing 50% DMF. The change of the fluorescence spectrum of the test solution was detected after 2 minutes of reaction, and the result is shown in FIG. 4. From fig. 4, it can be found that the fluorescence intensity of the test solution added with acetaldehyde, glyoxal, benzaldehyde, cysteine, glutathione, and glucose did not change significantly with respect to the blank test solution. However, the fluorescence intensity of the test solution added with formaldehyde was significantly enhanced. The experimental result shows that the near-infrared fluorescent polymer probe has good selectivity on formaldehyde.
Example 5: imaging of near-infrared fluorescent polymer probes in formaldehyde-containing cells
10. mu.L of the fluorescent probe stock solution obtained in example 2 was added to a HeLa cell-cultured dish (containing 1mM PBS medium) to give a probe concentration of 5. mu. mol/L, and incubated for 30 minutes to prepare two reference groups; the samples of one of the reference groups were added with 10. mu. mol/L of formaldehyde, and the incubation was continued for 20 minutes to obtain the experimental group. Then fluorescence imaging is performed on the reference group and the experimental group by a confocal microscope respectively, a light source with the excitation wavelength of 650nm is used for excitation, and the fluorescence in the range of 650-800nm is collected, and the result is shown in FIG. 5. In fluorescence imaging of the reference group, almost no fluorescence was observed; however, in the experimental group, a distinct red fluorescence was observed, with a significant increase in fluorescence. The experimental result shows that the near-infrared fluorescent polymer probe can detect formaldehyde in a cell environment through a confocal microscope, and has potential practical application value.
According to the near-infrared fluorescent polymer probe disclosed by the invention, when the near-infrared fluorescent polymer probe is exposed in a formaldehyde environment, a hydrazine group contained in the probe structure and formaldehyde are subjected to condensation reaction to generate hydrazone, so that the original photoelectron transfer (PET) effect disappears, and the squaraine recovers fluorescence.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (9)
1. A near-infrared fluorescent polymer probe for identifying formaldehyde, which is characterized in that the polymer probe has a general structure shown in formula I:
wherein n is 30-120.
2. The method for preparing the near-infrared fluorescent polymer probe for identifying formaldehyde as claimed in claim 1, characterized by comprising the steps of: mixing chitosan and near-infrared fluorescent molecules, and carrying out acylation reaction under the action of a combined catalyst to obtain the polymer probe containing the near-infrared fluorescent chitosan.
3. The method for preparing the near-infrared fluorescent polymer probe for identifying formaldehyde as claimed in claim 2, wherein the near-infrared fluorescent molecule has a structure of formula II:
4. the method for preparing the near-infrared fluorescent polymer probe for identifying formaldehyde as claimed in claim 2, wherein the mass parts of the chitosan and the near-infrared fluorescent molecules are as follows: 1-3 parts of chitosan and 5-10 parts of near-infrared fluorescent molecules.
5. The method for preparing a formaldehyde-recognizing near-infrared fluorescent polymer probe as claimed in claim 2, wherein the combined catalyst is N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide.
6. The method for preparing the near-infrared fluorescent polymer probe for identifying formaldehyde as claimed in claim 5, wherein the molar ratio of the N-hydroxysuccinimide, the near-infrared fluorescent molecule and the 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 1:1: 3-5.
7. The method for preparing the near-infrared fluorescent polymer probe for identifying formaldehyde as claimed in claim 2, wherein the acylation reaction is carried out at room temperature for 12-48 h.
8. The method for preparing the near-infrared fluorescent polymer probe capable of recognizing formaldehyde as claimed in claim 2, wherein the molecular weight Mw of the chitosan is 5-20 kDa, and the deacetylation degree is 0.8-0.95.
9. The use of the near-infrared fluorescent polymer probe for recognizing formaldehyde of claim 1 for specifically detecting formaldehyde in an aqueous solution.
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| CN110702746B (en) * | 2019-10-28 | 2021-12-10 | 苏州大学 | Condensed ring squaramide polymer, humidity-sensitive sensor based on condensed ring squaramide polymer and preparation method of humidity-sensitive sensor |
| CN113788821B (en) * | 2021-08-24 | 2022-06-14 | 陕西师范大学 | Near-infrared hydrazine compound, preparation method, formaldehyde detection kit and application |
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