CN110128566A - A kind of near-infrared fluorescent polymer probe and its preparation method and application identifying formaldehyde - Google Patents
A kind of near-infrared fluorescent polymer probe and its preparation method and application identifying formaldehyde Download PDFInfo
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- CN110128566A CN110128566A CN201910363791.7A CN201910363791A CN110128566A CN 110128566 A CN110128566 A CN 110128566A CN 201910363791 A CN201910363791 A CN 201910363791A CN 110128566 A CN110128566 A CN 110128566A
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- infrared fluorescent
- formaldehyde
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- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 title claims abstract description 209
- 239000000523 sample Substances 0.000 title claims abstract description 75
- 229920001109 fluorescent polymer Polymers 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 229920001661 Chitosan Polymers 0.000 claims abstract description 20
- 229920000642 polymer Polymers 0.000 claims abstract description 11
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 7
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical group ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 3
- 238000005917 acylation reaction Methods 0.000 claims description 3
- 239000003054 catalyst Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 230000003834 intracellular effect Effects 0.000 claims description 3
- 230000006196 deacetylation Effects 0.000 claims description 2
- 238000003381 deacetylation reaction Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000011895 specific detection Methods 0.000 claims 2
- 230000004044 response Effects 0.000 abstract description 8
- 238000006243 chemical reaction Methods 0.000 abstract description 4
- 229930040373 Paraformaldehyde Natural products 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 229920002866 paraformaldehyde Polymers 0.000 abstract description 3
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 238000003384 imaging method Methods 0.000 abstract 2
- 239000012452 mother liquor Substances 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 108010024636 Glutathione Proteins 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 229960003180 glutathione Drugs 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000011938 amidation process Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 229940015043 glyoxal Drugs 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 2
- -1 phenolic aldehyde Chemical class 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 108010028700 Amine Oxidase (Copper-Containing) Proteins 0.000 description 1
- XDQGMXYCBZNEAG-UHFFFAOYSA-N C(C)[C]CCCN(C)C Chemical compound C(C)[C]CCCN(C)C XDQGMXYCBZNEAG-UHFFFAOYSA-N 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102100027159 Membrane primary amine oxidase Human genes 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 229940095076 benzaldehyde Drugs 0.000 description 1
- 150000003935 benzaldehydes Chemical class 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000003930 cognitive ability Effects 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 229960002433 cysteine Drugs 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003760 hair shine Effects 0.000 description 1
- 125000000717 hydrazino group Chemical group [H]N([*])N([H])[H] 0.000 description 1
- 150000007857 hydrazones Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000037041 intracellular level Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000005976 liver dysfunction Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000003333 near-infrared imaging Methods 0.000 description 1
- 210000004126 nerve fiber Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 238000005424 photoluminescence Methods 0.000 description 1
- 231100000760 phototoxic Toxicity 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000006886 spatial memory Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- 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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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- 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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- 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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- 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"
- 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 kind of near-infrared fluorescent polymer probes and its preparation method and application for identifying formaldehyde, specifically disclose the near-infrared fluorescent polymer probe of structure shown in formula I.Polymer probe provided by the invention is formed by near-infrared fluorescent molecule and biocompatibility chitosan after chemical reaction, wherein, the near-infrared fluorescent molecule can occur chemical reaction with formaldehyde and issue feux rouges, realize the specificly-response of PARA FORMALDEHYDE PRILLS(91,95) as near-infrared fluorescent response unit；Main chain Component units of the chitosan as polymer assign polymer probe good water-soluble and biocompatibility.Near-infrared fluorescent polymer probe provided by the invention can be used in aqueous solution, in living cells and intravital formaldehyde examination and imaging, with important application prospects in terms of environmental monitoring, bio-imaging and sensing.
Description
Technical field
The invention belongs to high molecular materials and chemical sensor field, and in particular to a kind of near-infrared fluorescent for identifying formaldehyde
Polymer probe and its preparation method and application.
Background technique
Formaldehyde is a kind of colourless gas with specific stimulation smell, is confirmed to be and makees with strong carcinogenic and rush cancer
With to the easily exposure organ generation injury such as the eyes of people, nose, respiratory tract and skin.Currently, formaldehyde main source in living environment
In the metabolism and drop of the human production activities such as phenolic aldehyde and Lauxite, textile auxiliary agent and preservative and certain organic compounds
Solution.Formaldehyde is also human endogenous's property substance, can go first by semicarbazide-sensitive amine oxidase and lysine specific histone
The equal biological enzymes of base enzyme 1 generate.The study found that formaldehyde plays important work to the spatial memory of people and the formation of cognitive ability
With.However, will lead to the generation of some diseases, for example cancer, nerve fiber are degenerated, sugar when concentration of formaldehyde is exceeded in blood
Urine disease, senile dementia and chronic liver dysfunction etc..Therefore, it effectively monitors or monitors in biological sample or environmental sample
Formaldehyde has important practical significance.
Currently, some small-molecule fluorescent probes for formaldehyde examination have been reported.Patent CN201610077333 and specially
The fluorescence probe maximum emission wavelength that sharp CN2015109227960 is related to only 530nm, from ideal near infrared imaging window 650-
900nm gap is larger, this can bring photo-toxic to cell, can also induced tissue autofluorescence interference (the transmitting light of short wavelength
Exciting light as bioluminescence molecule in tissue)；Reported fluorescence probe is mostly non-water-soluble fluorescence small organic molecule,
These fluorescent small molecules are easy to be destroyed by biological enzyme in biotic environment, limit it in the fields such as life science, medicine
Using.Therefore, design, which synthesizes, is not only suitable for formaldehyde examination in solution environmental, and is suitable for intracellular levels of formaldehyde detection, and
And probe usage amount is few, the monitoring of safety and low toxicity, realization to intracellular formaldehyde, so that further auxiliary phase answers disease treatment and disease
The research of reason is the content that those skilled in the art need constantly to go to explore and create.
Summary of the invention
In order to overcome the above-mentioned defects in the prior art, the present invention is intended to provide a kind of near-infrared fluorescent for identifying formaldehyde is poly-
Close physical prospecting needle and its preparation method and application.Core of the invention is to utilize the sour cyanines near-infrared fluorescent in side and chitosan biological phase
Capacitive.Compared with small-molecule fluorescent probe, polymer probe of the invention is using chitosan polymer chain cheap and easy to get as base
Plinth is acted on using the collaborative combination of the sour cyanines recognition site in multiple hydrazines-side and neighbouring hydroxyl come enriching low-concentration formaldehyde pollutants.Cause
This, formaldehyde is reacted with the specific chemical of probe to be dramatically speeded up, and is shortened the fluorescence response time, is improved response sensitivity.
The first object of the present invention is to provide a kind of near-infrared fluorescent polymer probe for identifying formaldehyde, solves existing formaldehyde
Sensor cytotoxicity, stability and water solubility problems.The near-infrared fluorescent polymer probe of identification formaldehyde of the present invention,
Its general formula structure is formula I:
Formula I.
Second purpose of the invention is to provide the preparation method of the near-infrared fluorescent polymer probe of the identification formaldehyde, tool
Steps are as follows for body: using chitosan as main polymer chain, the sour cyanines derivative in side that identification formaldehyde is introduced by amidation process, that is, red
Outer fluorescent molecule obtains the chitosan derivatives containing the sour cyanines derivatives group in hydrazine-side.
Preferably, the near-infrared fluorescent molecule has II structure of formula:
Formula II.
Preferably, the mass fraction of the chitosan and near-infrared fluorescent molecule II are as follows: 1~3 part of chitosan, near-infrared is glimmering
II 5~10 parts of optical molecule.
Preferably, the molecular weight Mw of the chitosan is 5kDa~20kDa, and deacetylation is 0.8~0.95.
Preferably, the combination catalyst is N- hydroxysuccinimide and 1- ethyl-(3- dimethylamino-propyl) carbon two
Imines.
Preferably, the N- hydroxysuccinimide, near-infrared fluorescent molecule II and 1- ethyl-(3- dimethylamino-propyl)
The molar ratio of carbodiimide is 1:1:3-5.
Preferably, the acylation reaction carries out at room temperature, reaction time 12-48h.
Third object of the present invention is to provide a kind of near-infrared fluorescent polymer probes for identifying formaldehyde to examine in specificity
Survey the application of formaldehyde in aqueous solution.
Fourth object of the present invention be to provide it is a kind of identify formaldehyde near-infrared fluorescent polymer probe can specificity
Detect the application in endogenous cellular or exogenous formaldehyde.
Compared with the prior art, the advantages of the present invention are as follows:
1) the near-infrared fluorescent polymer probe of identification formaldehyde provided by the invention is biocompatibility, the shell in probe structure
Fraction ensure that the water solubility and cell compatibility of probe；
2) the near-infrared fluorescent polymer probe chromophore of identification formaldehyde provided by the invention is with chemical bonds in macromolecule
In, it is not easy to it falls off；
3) the near-infrared fluorescent polymer probe chromophore of identification formaldehyde provided by the invention is evenly distributed, and stable content shines
Performance and light guide are functional；
4) the near-infrared fluorescent polymer probe of identification formaldehyde provided by the invention, has good photostability, absorbs wave
A length of 663nm, launch wavelength 698nm have fluorescence within the scope of bio-safety near-infrared (NIR) window 650-900nm
The response of " on/off " type detects interference of the background to result, improves the accuracy of detection when can eliminate detection significantly；
5) " on/off " type formaldehyde probe PARA FORMALDEHYDE PRILLS(91,95) solution of squarylium type dyestuff provided by the invention has good response, energy
Enough detections realized to intracellular formaldehyde have many advantages, such as that low in cost, response is sensitive, easy to spread and applies.
Detailed description of the invention
Fig. 1 is the uv-visible absorption spectra of the near-infrared fluorescent polymer probe of identification formaldehyde of the invention；
Fig. 2 is 1 middle probe of the embodiment of the present invention with the situation of change of the addition fluorogram of different equivalent of formaldehyde, in figure, under
Supreme, concentration of formaldehyde is followed successively by the fluorescence spectrum of 0,2,4,6,8,10,12,16,32 μm of ol/L；
Fig. 3 is 1 middle probe of the embodiment of the present invention and 2 equivalent of formaldehyde in 1min, and fluorescence intensity level changes over time at 698 nm
Spectrogram；
Fig. 4 is selective column fluorescence data figure of 1 middle probe of the embodiment of the present invention to disturbance analyte；In figure, 1 half Guangs
Propylhomoserin, 2 glutathione, 3 acetaldehyde, 4 glyoxals, 5 benzaldehydes, 6 formaldehyde, 7 glucose；
Fig. 5 is 1 middle probe of the embodiment fluorescence imaging figure that formaldehyde responds in HeLa cell, wherein Fig. 5 (a) is reference group, 5
It (b) is experimental group.
Specific embodiment
Technical solution of the present invention is described further with reference to the accompanying drawing.
The near-infrared fluorescent polymer probe of identification formaldehyde of the invention, molecular structural formula such as formula I:
Formula I
Wherein, n=30~120.
The preparation method of the near-infrared fluorescent polymer probe of identification formaldehyde provided by the invention includes the following steps:
Using chitosan as main polymer chain, near-infrared fluorescent molecule is that can activate fluorescent functional molecule, passes through the ammonia of chitosan
Amidation process occurs for the carboxyl of base and near-infrared fluorescent molecule, introduced for chitosan have can specific recognition formaldehyde and
" unlatching " near-infrared fluorescent functional molecular obtains the near-infrared fluorescent polymer probe that can identify formaldehyde.
The near-infrared fluorescent molecule that the present invention uses has II structure of formula:
Formula II
Chitosan is native biopolymer, and source is extremely wide, safe and non-toxic, cheap, compared with synthesizing macromolecule, tool
There are good biocompatibility and biodegradability, therefore, using chitosan as raw material, polymer probe obtained is as solution
Or formaldehyde probe has very high environment friendly and biological safety in cell.
Embodiment 1 identifies the preparation of the near-infrared fluorescent polymer probe of formaldehyde
It takes 3.0g chitosan (Mw=10kDa, DA=0.85) to be dissolved in 25mLN, in N '-dimethylformamide (DMF), is protected in logical nitrogen
Shield is lower to be added the N- hydroxysuccinimide (NHS) of 5.0g near-infrared fluorescent molecule II and 5mL containing 0.01mol, adds again after stirring
Enter 0.04mol1- ethyl-(3- dimethylamino-propyl) carbodiimide (EDC), is stirred to react under room temperature for 24 hours.In deionization
Two days, vacuum freeze drying of dialysis, obtain target-probe, are put in ultraviolet absorption spectrum instrument and are tested, obtain Fig. 1 institute in water
Show uv-visible absorption spectra, it is seen that the maximum absorption band of target near infrared fluorescent probe in aqueous solution is located at 663nm.
2 near-infrared fluorescent polymer probe of embodiment changes from the fluorescence spectrum that different equivalent of formaldehyde react
Probe prepared by Example 1 is dissolved in DMF, and it is that (concentration of probe is 0.5 to 0.5mmol/L probe mother liquor that concentration, which is made,
Mmol/L)；Distilled water is added in the formalin that mass fraction is 37%, it is female to be configured to the formaldehyde that concentration of formaldehyde is 1mmol/L
Liquid.It is added in 5mL centrifuge tube from 60 μ L are taken out in probe mother liquor, the formaldehyde mother liquor of different equivalents (0-2eq) is added, it is described to work as
Amount be formaldehyde in formaldehyde mother liquor molal quantity relative to probe mother liquor middle probe molal quantity multiple, take 1.44 mLDMF and
The different volumes PBS aqueous solution of 25 mmol/L, pH 7.4 of concentration is diluted to 3mL, is configured to concentration and probe concentration and is 10 μm of ol/L, contains
The test solution of 50%DMF.(swashed using Fluorescence Spectrometer test probe from the variation of the fluorescence spectrum of different equivalent of formaldehyde reaction solutions
Hair wavelength is 650nm), as shown in Figure 2.From Figure 2 it can be seen that as gradually increasing for equivalent, near-infrared of the invention is added in formaldehyde
Photoluminescence peak of the fluorescent polymer probe solution at 698nm gradually increases.When fluorescence intensity reaches maximum value, than probe sky
The fluorescence intensity of white liquor enhances 7.4 times.
The above ultraviolet-ray visible absorbing and fluorescent emission test show absorption and the transmitted wave of the near infrared fluorescent probe
It is long to be located near infrared region.
3 near-infrared fluorescent polymer probe of embodiment and formaldehyde with different time change in fluorescence
It takes out 60 μ L from 2 fluorescence probe mother liquor of embodiment to be added in 5mL centrifuge tube, adding 60 μ L concentration is 0.5
The formaldehyde mother liquor of mmol/L, after stirring again with the PBS aqueous solution of 1.44mLDMF and 1.44mL (25 mmol/L of concentration,
PH7.4) be diluted to 3mL, be configured to concentration and probe concentration be 10 μm of ol/L, concentration of formaldehyde 3mmol/L, the test containing 50% DMF it is molten
Liquid.With the excitation wavelength of 650 nm, its fluorescence spectrum changed over time is tested, as a result as shown in Figure 3.As seen from Figure 3, with
Time increases, and the fluorescence intensity at 698nm becomes larger, and reaches maximum value at 30 seconds or so.
Selection Journal of Sex Research of the 4 near-infrared fluorescent polymer probe of embodiment to disturbance analyte
60 μ L are taken out from 2 fluorescence probe mother liquor of embodiment to be added in 5mL centrifuge tube, are separately added into following various concentration
Analyte: 20 μm of mol/L formaldehyde, 0.5mmol/L acetaldehyde, the glyoxal of 50 μm of ol/L, the benzaldehyde of 1mmol/L, cysteine,
Glutathione, glucose are diluted to the PBS aqueous solution (concentration 25mmol/L, pH 7.4) of 1.44mL DMF and different volumes
3mL, being configured to concentration and probe concentration is 10 μm of ol/L, the test solution containing 50% DMF.The fluorescence of test fluid is detected after reaction 2 minutes
Spectrum change, as a result as shown in Figure 4.By Fig. 4 it can be found that relative to skip test liquid, be added acetaldehyde, glyoxal, benzaldehyde,
Cysteine, glutathione, glucose test fluid fluorescence intensity there is no significant change.However, the test fluid of formaldehyde is added
Fluorescence intensity is significantly increased.It is good that experimental result illustrates that near-infrared fluorescent polymer probe PARA FORMALDEHYDE PRILLS(91,95) of the invention has
Selectivity.
Embodiment 5: near-infrared fluorescent polymer probe is imaged in cell containing formaldehyde
10 μ L are taken out from 2 fluorescence probe mother liquor of embodiment is added to the culture dish (culture medium containing 1mLPBS) for giving birth to HeLa cell
In, obtaining concentration and probe concentration is 5 μm of ol/L, is incubated for 30 minutes, prepares two parts of reference groups；In wherein one group of reference group sample respectively
The formaldehyde of 10 μm of ol/L is added, continues to be incubated for 20 minutes, as experimental group.Then respectively with Laser Scanning Confocal Microscope to reference group and
Experimental group carries out fluorescence imaging, and the light source activation for the use of excitation wavelength being 650nm collects the fluorescence of 650-800nm range, as a result
As shown in Figure 5.In the fluorescence imaging of reference group, it is barely perceivable fluorescence；However, in experimental group, it can be observed that bright
Aobvious red fluorescence, fluorescence significantly increase.Experimental result illustrates that near-infrared fluorescent polymer probe of the invention can be by altogether
Focusing microscope detects the formaldehyde in cellular environment, has potential practical application value.
Near-infrared fluorescent polymer probe of the invention is exposed under formaldehyde environment, the diazanyl and formaldehyde that probe structure contains
Generation condensation reaction generates hydrazone, so that original photoelectron transfer (PET) effect disappears and the sour cyanines recovery fluorescence in the side of making, passes through
Above scheme can get the fluorescence response of fluorescence " on/off " type, substantially increase the sensitivity of detection.
Taking the above-mentioned ideal embodiment according to the present invention as inspiration, through the above description, relevant staff is complete
Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention
Property range is not limited to the contents of the specification, it is necessary to which the technical scope thereof is determined according to the scope of the claim.
Claims (10)
1. a kind of near-infrared fluorescent polymer probe for identifying formaldehyde, which is characterized in that the polymer probe has the logical of formula I
Formula structure:
Wherein n=30~120.
2. the preparation method of the near-infrared fluorescent polymer probe of identification formaldehyde described in claim 1, which is characterized in that including
Following steps: by chitosan and near-infrared fluorescent molecular mixing, acylation reaction is carried out under combination catalyst effect, is obtained containing close
The polymer probe of IR fluorescence chitosan.
3. the preparation method of the near-infrared fluorescent polymer probe of identification formaldehyde described in claim 1, which is characterized in that described
Near-infrared fluorescent molecule has II structure of formula:
4. the preparation method of the near-infrared fluorescent polymer probe of identification formaldehyde according to claim 2, which is characterized in that
The mass fraction of the chitosan and near-infrared fluorescent molecule II are as follows: 1~3 part of chitosan, near-infrared fluorescent molecule II 5~10
Part.
5. the preparation method of the near-infrared fluorescent polymer probe of identification formaldehyde according to claim 2, which is characterized in that
The combination catalyst is N- hydroxysuccinimide and 1- ethyl-(3- dimethylamino-propyl) carbodiimide.
6. the preparation method of the near-infrared fluorescent polymer probe of identification formaldehyde according to claim 5, which is characterized in that
Mole of the N- hydroxysuccinimide, near-infrared fluorescent molecule II and 1- ethyl-(3- dimethylamino-propyl) carbodiimide
Than for 1:1:3-5.
7. the preparation method of the near-infrared fluorescent polymer probe of identification formaldehyde according to claim 2, which is characterized in that
The acylation reaction carries out at room temperature, and the reaction time is 12~48h.
8. the preparation method of the near-infrared fluorescent polymer probe of identification formaldehyde according to claim 2, which is characterized in that
The molecular weight Mw of the chitosan is 5kDa~20kDa, and deacetylation is 0.8~0.95.
9. a kind of near-infrared fluorescent polymer probe for identifying formaldehyde as described in claim 1 is in specific detection aqueous solution
The application of formaldehyde.
10. a kind of near-infrared fluorescent polymer probe for identifying formaldehyde as described in claim 1 is intracellular in specific detection
Application in source property or exogenous formaldehyde.
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