CN111875717B - Cyclodextrin type fluorescent probe and preparation method and application thereof - Google Patents

Cyclodextrin type fluorescent probe and preparation method and application thereof Download PDF

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CN111875717B
CN111875717B CN202010667179.1A CN202010667179A CN111875717B CN 111875717 B CN111875717 B CN 111875717B CN 202010667179 A CN202010667179 A CN 202010667179A CN 111875717 B CN111875717 B CN 111875717B
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cyclodextrin
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beta
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stirring
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CN111875717A (en
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王金
胡晓
卢斌
罗婉悦
周荣烨
张楠楠
何咏菁
李曼玉
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Yancheng Teachers University
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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
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • 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"
    • 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
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/14—Macromolecular compounds
    • C09K2211/1441—Heterocyclic
    • C09K2211/145—Heterocyclic containing oxygen as the only heteroatom
    • 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"
    • G01N2021/6432—Quenching

Abstract

The invention relates to a cyclodextrin fluorescent probe and a preparation method and application thereof.A beta-CD derivative with high activity is obtained by introducing aldehyde group into 6 position of beta-CD by two chemical synthesis methods and then condensed with a color development group thiosemicarbazide to obtain the characteristic of identifying Cu in aqueous solution2＋A fluorescence quenching probe. The probe is used for detecting the content of copper ions by fluorescence, overcomes the defect of low sensitivity of the existing method, has good identification capability on the copper ions, hardly generates interference on the copper ions by other ions, and has high sensitivity and specificity.

Description

Cyclodextrin type fluorescent probe and preparation method and application thereof
Technical Field
The invention relates to synthesis of a fluorescent probe, in particular to a cyclodextrin type fluorescent probe for detecting copper ions and a preparation method and application thereof.
Background
Cu2+The ions are essential trace elements for human body and participate in many biochemical reaction processes, and the biochemical function in the organism is mainly catalysis. Cu (copper)2+The ion has the following effects in human body: a component constituting a copper-containing enzyme and a copper-binding protein; maintain normal hematopoietic function, participate in iron metabolism and erythropoiesis; promoting connective tissue formation; protecting the central nervous system; promoting normal melanin formation and maintaining normal hair structure; protecting body cells from damage by superoxide anions; influence hormone secretion; the copper ion-free glucose-lowering health care wine has the beneficial effects that the metabolism of sugar and lipid is influenced, the glucose tolerance is reduced after copper is deficient, the condition of a patient is obviously improved by treating the patient with diabetes mellitus who is ineffective by conventional therapy with a small dose of copper ions, and the cholesterol level in blood is increased by reducing the blood sugar and lacking the copper. However, if the amount is excessive, the drug stays in the body and accumulates in various tissues, causing lesions and causing toxicity. When the copper metabolism of a human body is abnormal, Menkes disease and Alzheimer disease can be caused, and diseases such as liver and kidney injury can be caused. With Cu2+Is also one of the transition metal pollutants, so the research and detection of Cu2＋The fluorescence sensor has great significance to life sciences and environmental pollution.
At present, Cu2+The detection methods mainly comprise spectrophotometry, atomic absorption spectrometry, resonance scattering spectrometry, electrochemistry and the like, but the methods have the defects of large interference, poor sensitivity, expensive instruments and the like, and the methods need to be operated by professional personnel and have high cost. Therefore, Cu with simple development operation and low cost2+The detection method has important application value.
Disclosure of Invention
The invention aims to provide a cyclodextrin fluorescent probe, a preparation method and application thereof, so as to achieve the purpose of improving the sensitivity and specificity of fluorescence detection of copper ions.
In order to solve the above technical problems, according to an aspect of the present invention, there is provided a cyclodextrin-type fluorescent probe, in which an thiosemicarbazide group is introduced at the 6-position of β -cyclodextrin, and the structural formula of the probe is:
Figure 199200DEST_PATH_IMAGE002
wherein R is selected from H, CH3、C2H5Ph, Bn or a heterocycle.
According to another aspect of the present invention, there is provided a method for preparing the above cyclodextrin type fluorescent probe, comprising:
introducing aldehyde group into 6 site of beta-cyclodextrin to obtain cyclodextrin aldehyde;
dissolving the cyclodextrin aldehyde and the thiosemicarbazide obtained in the step one in absolute methanol, and stirring at the temperature of 70-80 ℃ for reaction; and after the reaction is finished, when the temperature of the reaction liquid is reduced to 20-30 ℃, separating out solids, carrying out vacuum filtration to obtain a filter cake, washing the filter cake with absolute ethyl alcohol, and drying the washed product to obtain the cyclodextrin type fluorescent probe.
Preferably, in the second step, after the cyclodextrin aldehyde and the thiosemicarbazide are dissolved in the anhydrous methanol, stirring and reacting at the temperature of 75 ℃; after the reaction, the temperature of the reaction solution was decreased to 25 ℃ and a solid was precipitated.
Further, in step one, a first process for obtaining cyclodextrin aldehydes, comprising the steps of:
dissolving beta-cyclodextrin in NaOH solution, adding tosyl chloride into the solution, and stirring for reaction; after the reaction is completed, carrying out suction filtration, adjusting the pH of the filtrate to 7 by hydrochloric acid, stirring, and carrying out suction filtration to obtain a white precipitate; dissolving the white precipitate in deionized water, stirring and heating to 80-90 deg.C, and filtering to remove a small amount of white insoluble solid; cooling and recrystallizing the filtrate to separate out a large amount of white precipitates, and performing suction filtration to obtain a crude product, namely the mono-6-benzenesulfonyl-beta-cyclodextrin;
weighing the obtained mono-6-benzenesulfonyl-beta-cyclodextrin, dissolving in dimethyl sulfoxide (DMSO) in N2Under protection, the temperature is raised to 130-140 ℃ and the reaction is stirred; dropwise adding the obtained reddish brown reaction liquid into ethyl acetate, separating out brown precipitate, and filtering to remove filtrate; and then vacuum drying is carried out, and the obtained product is the cyclodextrin aldehyde.
Preferably, the white precipitate is dissolved in deionized water and heated to 85 ℃ with stirring.
Preferably, a sheet-6-benzenesulfonyl-beta-cyclodextrin in dimethyl sulfoxide in N2The temperature was raised to 135 ℃ with stirring and reacted for 16 h under protection.
Further, in step one, a second process for obtaining cyclodextrin aldehydes, comprising the steps of:
weighing beta-cyclodextrin and IBX, dissolving in dimethyl sulfoxide, placing in a dry and sealed round-bottom flask, stirring for reaction, and filtering to remove a filter cake; dripping the filtrate into ethyl acetate to separate out white precipitate, continuously stirring, and performing vacuum filtration to remove the filtrate; adding deionized water into the white precipitate to dissolve, and filtering to remove white insoluble solid; and (4) performing vacuum freeze drying to obtain a white powder solid, wherein the obtained product is the cyclodextrin aldehyde.
Preferably, the reaction is stirred in a dry closed round bottom flask at 25 ℃ for 24 h.
According to another aspect of the present invention, there is provided the use of the cyclodextrin-type fluorescent probe for identifying copper ions.
The invention adopts two chemical synthesis methods to introduce aldehyde group at the 6-position of beta-CD to obtain beta-CD derivative with high activity, and then the beta-CD derivative is condensed with chromophoric group thiosemicarbazide to obtain the product for identifying Cu in aqueous solution2＋A fluorescence quenching probe. The probe is used for detecting the content of copper ions by fluorescence, overcomes the defect of low sensitivity of the existing method, has good identification capability on the copper ions, hardly generates interference on the copper ions by other ions, and has high sensitivity and specificity.
Drawings
FIG. 1 shows the probe L prepared in example 3 of the present invention for Cu among other cations2+Selective identification of (2);
FIG. 2 shows the probe L prepared in example 3 of the present invention for Cu among other anions2+Selective identification of (2);
FIG. 3 shows Cu of probe L prepared in example 3 of the present invention2+Titration curve chart;
FIG. 4 is a measurement of a working curve of a probe L prepared in example 3 of the present invention;
FIG. 5 shows probes L and Cu prepared in example 3 of the present invention2＋Experiment of binding ratio；
FIG. 6 shows the probe L prepared in example 3 of the present invention for Cu among other cations2＋Interference analysis of (3);
FIG. 7 shows the probe L prepared in example 3 of the present invention for Cu among other anions2＋Interference analysis of (4).
Detailed Description
The invention provides a cyclodextrin fluorescent probe, which introduces thiosemicarbazide group at 6-position of beta-cyclodextrin (beta-CD), and the structural formula is as follows:
Figure 490242DEST_PATH_IMAGE002
wherein R is selected from H, CH3、C2H5Ph, Bn or a heterocycle.
The cyclodextrin fluorescent probe can be used for detecting the content of copper ions, can overcome the defect of low sensitivity of the existing method, has good identification capability on the copper ions, hardly generates interference on the copper ions by other ions, and has high sensitivity and specificity. Cyclodextrins are a class of cyclic oligosaccharides that encapsulate various organic or biological molecules within a hydrophobic cavity, and such oligosaccharides are water soluble and biocompatible. The cyclodextrin has a special structure that the outer wall is hydrophilic and the inner cavity is hydrophobic, and can form coordination with metal ions in aqueous solution to enhance a fluorescence signal.
Another exemplary embodiment of the present invention provides a method for preparing the cyclodextrin-type fluorescent probe described above, which includes:
introducing aldehyde group into 6 site of beta-cyclodextrin to obtain cyclodextrin aldehyde;
dissolving the cyclodextrin aldehyde and the thiosemicarbazide obtained in the step one in absolute methanol, and stirring at the temperature of 70-80 ℃ for reaction; and after the reaction is finished, when the temperature of the reaction liquid is reduced to 20-30 ℃, separating out solids, carrying out vacuum filtration to obtain a filter cake, washing the filter cake with absolute ethyl alcohol, and drying the washed product to obtain the cyclodextrin type fluorescent probe. Preferably, in the second step, after the cyclodextrin aldehyde and the thiosemicarbazide are dissolved in the anhydrous methanol, the reaction is stirred at the temperature of 75 ℃; after the reaction, the temperature of the reaction solution was decreased to 25 ℃ and a solid was precipitated.
The thiosemicarbazide in the second step comprises substituted or unsubstituted thiosemicarbazide, and R is selected from H, CH3、C2H5Ph, Bn or a heterocycle. Taking unsubstituted thiosemicarbazide as an example, the synthetic route is as follows:
Figure 597875DEST_PATH_IMAGE004
the basic concept of the preparation method is to introduce aldehyde group at the 6-position of beta-CD to obtain beta-CD derivative with high activity, and then condense the beta-CD derivative with chromophoric group thiosemicarbazide to design the method capable of identifying Cu in aqueous solution2＋A fluorescence quenching probe. By passing1The probe is structurally characterized by H-NMR, FTIR and CD, and is researched by a fluorescence spectrum titration experiment, a Job's curve and an ion interference test under a Tris-HCl and pH 7.4 system, so that the probe can specifically recognize Cu2＋Ions.
The invention provides two methods for synthesizing cyclodextrin aldehyde.
A first process for synthesizing cyclodextrin aldehydes comprising the steps of:
dissolving beta-cyclodextrin in NaOH solution, adding p-toluenesulfonyl chloride into the solution, wherein the molar ratio of beta-CD to p-toluenesulfonyl chloride is 1:0.6-1:1.8, and stirring for reaction; after the reaction is completed, carrying out suction filtration, adjusting the pH of the filtrate to 7 by hydrochloric acid, stirring, and carrying out suction filtration to obtain a white precipitate; dissolving the white precipitate in deionized water, stirring and heating to 80-90 deg.C, preferably to 85 deg.C. Filtering to remove a small amount of white insoluble solid; cooling and recrystallizing the filtrate to separate out a large amount of white precipitates, and performing suction filtration to obtain a crude product, namely the mono-6-benzenesulfonyl-beta-cyclodextrin.
Weighing the obtained mono-6-benzenesulfonyl-beta-cyclodextrin, dissolving in dimethyl sulfoxide (DMSO) in N2Under protection, the temperature is raised to 130-140 ℃ and the reaction is stirred, preferably, the temperature is raised to 135 ℃ and the reaction is stirred for 16 h. Dropwise adding the obtained reddish brown reaction liquid into ethyl acetate, separating out brown precipitate, and filtering to remove filtrate;and then vacuum drying is carried out, and the obtained product is the cyclodextrin aldehyde.
Aiming at the method, the synthetic route of the cyclodextrin type fluorescent probe is as follows (R is H):
Figure DEST_PATH_IMAGE006
。
a second process for the synthesis of cyclodextrin aldehydes comprising the steps of:
weighing beta-cyclodextrin and IBX, dissolving in dimethyl sulfoxide, wherein the molar ratio of beta-CD to IBX is 1:1-1:2, placing in a dry and sealed round bottom flask, and stirring for reaction, preferably stirring for reaction at 25 ℃ for 24 h. Filtering to remove filter cakes; dripping the filtrate into ethyl acetate to separate out white precipitate, continuously stirring, and performing vacuum filtration to remove the filtrate; adding deionized water into the white precipitate to dissolve, and filtering to remove white insoluble solid; and (4) performing vacuum freeze drying to obtain a white powder solid, wherein the obtained product is the cyclodextrin aldehyde.
The technical solution and the technical effects claimed by the present invention will be further clearly and completely explained by some specific examples.
Example 1: preparation of Cyclodextrin aldehyde
Weighing 25g of beta-CD in 250 mL of NaOH solution, stirring to dissolve the beta-CD, slowly stirring and adding 17.5 g of paratoluensulfonyl chloride into the solution under the ice bath condition, and after the addition is finished, violently stirring and reacting for 1 h. And then, removing white impurities in the reaction solution by suction filtration, adjusting the pH of the filtrate to 7 by using 2M hydrochloric acid, continuously stirring for 1 h under the ice bath condition, wherein a large amount of white precipitates appear in the process, and obtaining the white precipitates by suction filtration. The white precipitate is dissolved in 340 mL of deionized water, heated to 85 ℃ with stirring, and filtered to remove a small amount of white insoluble solid when the solution is clear and transparent and hot. Cooling the filtrate to about 25 ℃, recrystallizing for 24 h (4 ℃), separating out a large amount of white precipitate, and filtering to obtain the white precipitate, namely the crude product. And (3) repeating the recrystallization step twice for purification to obtain a white solid, namely the mono-6-benzenesulfonyl-beta-cyclodextrin.
2.2 g of beta-CD-OTS are weighed out and dissolved in 18 mL of dimethyl sulfoxide, 4.5 mL of triethylamine are added as catalyst. In N2The temperature was raised to 135 ℃ with stirring for 16 h under protection (TLC assay with isopropanol/water/ammonia in 6:3:1 developing solvent, iodine vapor method). The color of the reaction liquid gradually changes from light yellow to reddish brown. Slowly dripping the reddish brown reaction liquid into 200 mL ethyl acetate to separate out a large amount of brown precipitate, continuously stirring for 15 min, and filtering to remove the filtrate. The brown precipitate was washed 3 to 4 times with absolute ethanol and subsequently dried in vacuo for 24 h (40 ℃). The obtained product is the cyclodextrin aldehyde.
Example 2: preparation of Cyclodextrin aldehyde
0.5 g of beta-CD was weighed out and dissolved in 4 mL of dimethyl sulfoxide, and 0.161 g of IBX was weighed out and dissolved in 6 mL of dimethyl sulfoxide. Placing the two into a dry and sealed round-bottom flask, stirring for reaction at 25 ℃ for 24 h (during the reaction, detecting by TLC, developing by using isopropanol/water/ammonia water as a developing agent, and developing by an iodine vapor method), and filtering to remove a filter cake. The filtrate was slowly added dropwise to 100 mL ethyl acetate to precipitate a large amount of white precipitate, stirring was continued for 10 min, and the filtrate was filtered off under reduced pressure. A small amount of deionized water was added to the white precipitate to dissolve it, and the insoluble white solid was removed by suction filtration. And (4) carrying out vacuum freeze drying on the filtrate for 48 hours to obtain a white powder solid, wherein the obtained product is the cyclodextrin aldehyde.
Example 3: cyclodextrin type fluorescent probe L
0.5 g of beta-CD-CHO and 60.3 mg of thiosemicarbazide were dissolved in 10 mL of anhydrous methanol. Mixing the two at 75 deg.C, stirring with magnetic stirrer, reacting, and refluxing for 12 hr. At first, a large amount of insoluble substances exist in the reaction liquid, the reaction liquid is yellow clear transparent solution when the temperature is raised to about 45 ℃, and golden yellow solid is separated out above the contact surface of the liquid surface and the wall of the round-bottom flask after the reaction is carried out for 30 min. After the reaction is finished, when the temperature of the reaction liquid is reduced to 25 ℃, solid is separated out and the color is light yellow. The filter cake was collected by suction filtration under reduced pressure and washed with anhydrous ethanol 3 to 4 times (detection by TLC, developing solvent isopropanol/water/ammonia water: 6:3:1, color development by iodine vapor method). The light yellow product was dried under vacuum at 40 ℃ for 24 h.
Example 4
The difference from example 1 is that: filtering to obtain white precipitate, dissolving the white precipitate in 340 mL deionized water, stirring and heating to 80 ℃, and filtering to remove a small amount of white insoluble solid when the white precipitate is clear and transparent and hot.
2.2 g of beta-CD-OTS were weighed out and dissolved in 18 mL of dimethyl sulfoxide, and 4.5 mL of triethylamine was added as a catalyst. In N2Under protection, the temperature is raised to 130 ℃ and the reaction is stirred for 16 h (TLC detection reaction, developing agent is isopropanol/water/ammonia water ═ 6:3:1, iodine vapor method color development).
Example 5
The difference from example 1 is that the white precipitate is obtained by suction filtration, dissolved in 340 mL of deionized water, heated to 90 ℃ with stirring, and filtered to remove a small amount of white insoluble solid when it is clear and transparent while it is hot.
2.2 g of beta-CD-OTS were weighed out and dissolved in 18 mL of dimethyl sulfoxide, and 4.5 mL of triethylamine was added as a catalyst. In N2Under protection, the temperature is raised to 140 ℃ and the reaction is stirred for 16 h (TLC detection reaction, developing agent is isopropanol/water/ammonia water ═ 6:3:1, iodine vapor method color development).
Example 6
The difference from example 3 is that: 0.5 g of beta-CD-CHO and 60.3 mg of thiosemicarbazide were dissolved in 10 mL of anhydrous methanol. Mixing the two at 70 deg.C, stirring with magnetic stirrer, and refluxing for 12 hr. At first, a large amount of insoluble substances exist in the reaction liquid, the reaction liquid is yellow clear transparent solution when the temperature is raised to about 45 ℃, and golden yellow solid is separated out above the contact surface of the liquid surface and the wall of the round-bottom flask after the reaction is carried out for 30 min. After the reaction is finished, when the temperature of the reaction liquid is reduced to 20 ℃, solid is separated out and the color is light yellow.
Example 7
The difference from example 3 is that: 0.5 g of beta-CD-CHO and 60.3 mg of thiosemicarbazide were dissolved in 10 mL of anhydrous methanol. Mixing the two at 80 deg.C, stirring with magnetic stirrer, reacting, and refluxing for 12 hr. At first, a large amount of insoluble substances exist in the reaction liquid, the reaction liquid is yellow clear transparent solution when the temperature is raised to about 45 ℃, and golden yellow solid is separated out above the contact surface of the liquid surface and the wall of the round-bottom flask after the reaction is carried out for 30 min. After the reaction is finished, when the temperature of the reaction liquid is reduced to 30 ℃, solid is separated out and the color is light yellow.
When R is selected from CH3、C2H5Ph, Bn or a heterocycle, the corresponding cyclodextrin-type fluorescent probe L can be prepared in the same manner as in example 3.
Next, taking as an example the cyclodextrin type fluorescent probe L (hereinafter referred to simply as probe L) obtained in example 3, each test for the fluorescent detection of copper ions by the probe L will be described.
Test method
Probe L Selectivity test
To 2 ml of the Tris-HCl buffer solution, 40. mu.L of probe L (1 eq) was added, and then 160. mu.L of each metal ion (4 eq) or each anion (4 eq) was added, respectively, to test the fluorescence spectrum of each sample after each ion was added.
2＋Cu fluorescence titration test
Adding 20 mu L of probe L solution into 2 mL of prepared Tris-HCl buffer solution, and measuring the fluorescence spectrum after uniformly mixing. Followed by 1. mu.L each addition of Cu2＋The solution was mixed well and tested each time Cu was added2＋Fluorescence spectrum of the latter sample, probe L vs. Cu was plotted2+And detecting a limit graph.
2＋Probe L/Cu binding ratio test
Adding a certain amount of probe L solution and Cu into 2 ml Tris-HCl buffer solution2＋Solution of probes L and Cu added thereto2＋Total concentration of (2) was fixed to 2.0X 10-5M, changing the molar ratio of the two (n)L : nCu 2＋9: 1; 8: 2; 7: 3; 6: 4; 5: 5; 4: 6; 3: 7; 2: 8; 1: 9; 0: 10), testing the ultraviolet spectrum of each proportion, and drawing a Job's curve graph.
Other cation interference test
Add 40. mu.L of Probe L (1 eq) to 2 mL of prepared Tris-HCl buffer solution, add 160. mu.L of other cations (4 eq) first, and add 160. mu.L of Cu2＋(4 eq), test each test runFluorescence spectrum of the sample.
Anion interference test
Add 40. mu.L of Probe L (1 eq) to 2 mL of prepared Tris-HCl buffer solution, add 160. mu.L of anions (4 eq) separately, and add 160. mu.L of Cu2＋(4 eq), the fluorescence spectrum of each sample was measured.
(II) test results
Ion selective recognition analysis
The fluorescence sensing selectivity of probe L was studied by comparing its fluorescence response to various metal ions. Here, the influence of an excessive amount of each metal ion at the same concentration was examined by detecting the change in fluorescence intensity of the probe L at an excitation wavelength of 380 nm. As shown in FIG. 1, Cu2+Shows 10.6-fold fluorescence quenching of the fluorescence of probe L. And probe L for Ca addition2＋，Mg2＋，Mn2＋，Cd2＋，Ba2＋，K＋，Na＋，Li＋After metal ions, a weak quenching phenomenon is exhibited. Adding Zn2＋After that, there was no significant change in the probe L. Adding Al3＋After that, the fluorescence of the probe L slightly increases. Cr (chromium) component3+，Ni2＋，Co2＋，Sn＋The addition of these four metal ions quenches the fluorescence of probe L, but far less than Cu2+The resulting effect is large. From the above, it can be concluded that Cu is contained in each metal ion2+The cation can cause better quenching phenomenon of the probe L, and other cations have no obvious effect on the probe L.
The fluorescence sensing selectivity of probe L was studied by comparing the fluorescence response of probe L to various anions. Here, the effect of excess of each anion at the same concentration was tested by detecting the change in fluorescence intensity of probe L at an excitation wavelength of 380 nm. As shown in FIG. 2, probes L vs. Cu2+Showing a 10.6-fold degree of fluorescence quenching. Adding SO4 2－，SO3 2－，HPO4 2－，CO3 2－，NO3 －，H2PO4 －，HSO4 －，HCO3 －，CH3COO－Probe L showed a slight quenching or a slight enhancement. In conclusion, it can be found that the probe L has no specific recognition effect on these anions, and is capable of detecting Cu2+Has strong recognition.
Fluorescence titration
Study of analytical probes L vs Cu by fluorescence titration2+Sensitivity of fluorescence quenching effect is generated, the concentration of the probe L is kept constant, and Cu is gradually increased2+The concentration of (a) and the fluorescence intensity thereof also change. As shown in FIG. 3, accompanying Cu2+The maximum emission wavelength of the system is gradually shifted and blue-shifted from 485 nm to 475 nm. The system has prominent fluorescence quenching change when Cu2+When the concentration reaches half of the concentration of the probe L, the fluorescence quenching effect tends to be slow. When the concentration of both is close to 1:1, the fluorescence intensity of the probe L of this system slightly decreases. Cu is visible under ultraviolet rays and the like2+The yellow fluorescence of the original probe gradually becomes dim until no fluorescence is generated. This indicates that probes L and Cu2+Possible 1:1 coordination, while also indicating probe L to Cu2+Can produce excellent fluorescence quenching effect.
Ion interference analysis
Probe L and Cu2+To analyze the Cu detection of the probe L by other metal ions by comparing the fluorescence sensing capability of other cation mixtures2+Because a very important feature of a fluorescence sensor is that its response to the analyte is the same as its response to other substances in the environment. As shown in FIG. 6, probe L also showed a slightly higher fluorescence quenching factor (F/F) in Tris-HCl (10 mM, pH = 7.4) system0= 0.21), wherein the majority is Cu alone2+（F/F0= 0.09) fluorescence quenching intensity similar buffer solution (K)+，Na+) Transition metal ion (Ni)2+，Mn2+，Co2+，Cd2+，Li＋) Alkaline earth metal ion (Ca)2+，Mg2+) And the like. Description of the inventionOther cation to Cu2+The interference of fluorescence quenching intensity is not large.
The probe L shows better specificity after other cation interference tests are carried out. At the same time, we will mix the probe L with Cu2+To analyze the recognition of Cu by the anion pair probes L2+The interference of (2). The laboratory now possesses 9 anions as in fig. 7. These anions were measured for fluorescence intensity in the same fluorescence spectrum measurement system as the above cations. As shown in FIG. seven, we can easily observe these 9 anions with Cu alone2+（F/F0= 0.09) fluorescence quenching intensity was also similar. The 9 common anion pairs Cu are also illustrated2+The interference of fluorescence quenching intensity is not large. In conclusion, we have successfully prepared a probe capable of specifically recognizing Cu in aqueous solution2+The fluorescent probe of (1).
Determination of the working Curve
According to probe L to Cu2+The fluorescence titration data of (1) was calculated for probe L vs Cu by computational fluorescence spectroscopy2+The detection limit of (2) was Tris-HCl (10 mM, pH = 7.4) as solvent system, the excitation wavelength was 380 nm, and the slit width (excitation and emission) was 5 nm/5 nm. First, a solution of the probe L was continuously scanned 10 times, and the fluorescence emission intensity at 470 nm thereof was measured. Then, a low equivalent continuous titration experiment was performed to obtain the fluorescence emission spectrum. A curve was obtained by plotting the fluorescence emission intensity at 470 nm against the concentration of Cu2+ (FIG. 4), and was fitted to it by its curve equation F =800.72936-11.46869[ Cu2+ ]2+] + 0.06446[Cu2+]2R = 0.98662. As shown in the fourth graph, the detection range was calculated to be 0 to 85. mu.M, and the detection limit was calculated to be 1.37. mu.M, indicating that the detection of Cu by the probe L pair was carried out2+Has better sensitivity.
Experiment of binding ratio
To further study probes L and Cu2+The coordination relationship between the probes L and Cu is determined by using an equimolar continuous variation method2+The interaction between themThe probes L and Cu were measured by Job's plot method after analysis2+The binding ratio of (A) and (B) is shown in FIG. 5, and when the molar ratio of probe L is close to 0.5, a turning point appears in the fluorescence emission intensity, indicating that the probe L and Cu are present2+The stoichiometric ratio between is 1:1.

Claims (4)

1. A cyclodextrin-type fluorescent probe characterized in that: introducing an aminosulfur urea group at the 6-position of beta-cyclodextrin, wherein the structural formula is as follows:
Figure DEST_PATH_IMAGE002
wherein R is selected from H, CH3、C2H5Ph, Bn or a heterocycle.
2. A method of preparing a cyclodextrin-type fluorescent probe of claim 1, comprising:
introducing aldehyde group into 6 site of beta-cyclodextrin to obtain cyclodextrin aldehyde;
a first process for obtaining cyclodextrin aldehydes comprising the steps of:
dissolving beta-cyclodextrin in NaOH solution, adding tosyl chloride into the solution, and stirring for reaction; after the reaction is completed, carrying out suction filtration, adjusting the pH of the filtrate to 7 by hydrochloric acid, stirring, and carrying out suction filtration to obtain a white precipitate; dissolving the white precipitate in deionized water, stirring and heating to 80-90 deg.C, and filtering to remove a small amount of white insoluble solid; cooling and recrystallizing the filtrate to separate out a large amount of white precipitates, and performing suction filtration to obtain a crude product, namely the mono-6-benzenesulfonyl-beta-cyclodextrin;
weighing the obtained mono-6-benzenesulfonyl-beta-cyclodextrin, dissolving in dimethyl sulfoxide (DMSO) in N2Under protection, the temperature is raised to 130-140 ℃ and the reaction is stirred; dropwise adding the obtained reddish brown reaction liquid into ethyl acetate, separating out brown precipitate, and filtering to remove filtrate; then vacuum drying is carried out, and the obtained product is cyclodextrin aldehyde;
a second process for obtaining cyclodextrin aldehydes comprising the steps of:
weighing beta-cyclodextrin and IBX, dissolving in dimethyl sulfoxide, placing in a dry and sealed round-bottom flask, stirring for reaction, and filtering to remove a filter cake; dripping the filtrate into ethyl acetate to separate out white precipitate, continuously stirring, and performing vacuum filtration to remove the filtrate; adding deionized water into the white precipitate to dissolve, and filtering to remove white insoluble solid; vacuum freeze drying to obtain white powder solid as cyclodextrin aldehyde;
dissolving the cyclodextrin aldehyde and the thiosemicarbazide obtained in the step one in absolute methanol, and stirring at the temperature of 70-80 ℃ for reaction; and after the reaction is finished, when the temperature of the reaction liquid is reduced to 20-30 ℃, separating out solids, carrying out vacuum filtration to obtain a filter cake, washing the filter cake with absolute ethyl alcohol, and drying the washed product to obtain the cyclodextrin type fluorescent probe.
3. The method of claim 2, wherein: in the second step, cyclodextrin aldehyde and thiosemicarbazide are dissolved in absolute methanol and stirred for reaction at the temperature of 75 ℃; after the reaction, the temperature of the reaction solution was decreased to 25 ℃ and a solid was precipitated.
4. Use of the cyclodextrin-type fluorescent probe of claim 1 for identifying copper ions.
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