CN109232589B - Fluorescent probe and preparation and application thereof - Google Patents
Fluorescent probe and preparation and application thereof Download PDFInfo
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- CN109232589B CN109232589B CN201811222729.8A CN201811222729A CN109232589B CN 109232589 B CN109232589 B CN 109232589B CN 201811222729 A CN201811222729 A CN 201811222729A CN 109232589 B CN109232589 B CN 109232589B
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Abstract
The invention discloses a fluorescent probe and preparation and application thereof. The probe is prepared from eight-membered cucurbituril (Q8) and acridine hydrochloride. The preparation process includes dissolving Q8 in water to obtain solution A; taking acridine hydrochloride, and dissolving the acridine hydrochloride in water to obtain a solution B; and mixing the solution A and the solution B, and regulating the pH value of the mixture to be 4 by using concentrated hydrochloric acid at normal temperature to prepare the probe. The application of the reagent is mainly used for detecting basic L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in water. The probe can detect alkaline L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in water, and has the characteristics of higher sensitivity, lower detection cost, simple sample treatment, convenient operation, quick determination and real-time detection.
Description
Technical Field
The invention relates to a fluorescent probe and preparation and application thereof, in particular to a fluorescent probe for detecting alkaline L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in water, and a preparation method and application thereof.
Background
Amino acids play an extremely important role in the growth and development of organisms: 1. is a constituent of human tissue; 2. various substances constituting the human body; 3. supplying heat; 4. immune regulation; 5. as an important carrier in vivo, plays a role in transportation; 6. and (4) an oxidation function. The amino acid is the basic composition substance of the protein, the intake of the amino acid is the only way for the human body to obtain the nitrogen source, after the human body absorbs the amino acid, a part of the amino acid is directly used for synthesizing the protein, a part of the amino acid is oxidized and decomposed, the nitrogen-containing part of the amino acid is used for synthesizing other necessary amino acid, and a part of the amino acid is decomposed as energy and is discharged out of the body in the form of urea.
Therefore, the method is particularly important for detecting the content and the type of amino acids in food, and the fluorescent probe is a novel detection reagent which is favored by people due to the advantages of higher sensitivity, lower detection cost, simple sample treatment, convenient operation, quick determination and real-time detection. It is necessary to develop a novel fluorescent probe for detecting L-amino acid in water and a novel fluorescent probe detection method.
Disclosure of Invention
The invention aims to provide a fluorescent probe and preparation and application thereof. The probe can detect alkaline L-amino acids (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in water, one probe can detect a plurality of amino acids, the detection of a single probe on a plurality of target amino acids can be realized, the detection cost is low, the detection efficiency is high, and the analysis of a complex microsystem is facilitated. In addition, when the probe is used for detection, the probe also has the characteristics of higher sensitivity, simple sample treatment, convenient operation, quick determination and real-time detection.
The technical scheme of the invention is as follows: a fluorescent probe, the molecular formula of the probe is C48H48N32O16@C13H10NCl。
A process for preparing the said fluorescent probe is prepared from eight-membered cucurbituril (Q8) and acridine hydrochloride.
In the above method for preparing fluorescent probe, the molar ratio of Q8 to acridine hydrochloride is 1: 0.5-2.
In the above method for preparing fluorescent probe, the molar ratio of Q8 to acridine hydrochloride is 1: 1.
the preparation method of the fluorescent probe specifically comprises the following steps:
(1) taking Q [8]]，Q[8]Dissolving in water to obtainSolution A at a concentration of 1.0 x 10-4mol/L；
(2) Dissolving acridine hydrochloride in water to obtain solution B with concentration of 1.0 × 10-3mol/L；
(3) Mixing solution A and solution B according to the weight ratio of 10: 1, namely mixing in a molar ratio of 1:1, reacting at normal temperature, and adjusting the pH value to be 4 to obtain the probe.
In the preparation method of the fluorescent probe, the water added in the step (1) and the step (2) is; secondary water with pH 7, and concentrated hydrochloric acid to adjust pH 4.
The application of the fluorescent probe is used for detecting various L-amino acids in water.
In the application of the fluorescent probe, the detection of various L-amino acids in water is performed; used for detecting alkaline L-amino acid, L-phenylalanine and L-tryptophan in water; the basic L-amino acids are L-arginine, L-histidine and L-lysine.
In the application of the fluorescent probe, when the fluorescent probe is used for detecting basic L-amino acid, L-phenylalanine and L-tryptophan in water, the specific detection method comprises the following steps:
1) diluting the fluorescent probe with pH 4 water to 2.0 x 10-5Obtaining a probe standard solution by using a mol/L solution;
2) adding water to be detected into the probe standard solution prepared in the step 1), standing for 10-20min, then performing fluorescence emission spectrometry at a fixed excitation wavelength of 346nm, and drawing a change curve of the fluorescence intensity at the excited laser wavelength;
3) and (3) calculating the fluorescence emission spectrum intensity change value delta F corresponding to 478nm before and after the water to be detected is added into the fluorescent probe solution according to the curve of the step 2), and detecting the basic L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in the water.
In the application of the fluorescent probe, in the step 3), when the fluorescence emission spectrum intensity corresponding to 478nm changes or undergoes blue shift before and after the water to be detected is added, it indicates that the water to be detected contains basic L-amino acids (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan; when the fluorescence emission spectrum intensity of the sample before and after the sample is added is not changed or blue shift does not occur corresponding to the fluorescence emission spectrum intensity under 478nm, the sample is indicated that the sample does not contain basic L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan.
In order to verify the beneficial effects of the invention, the inventor carries out a great deal of experimental research, and part of the experimental processes and results are as follows:
experimental example 1 an appropriate molar ratio of the probe formed by Q8 and acridine hydrochloride was investigated
In order to explore the proper molar ratio of the probe formed by Q8 and acridine hydrochloride, the interaction between the host and the object is investigated by ultraviolet absorption spectrometry and fluorescence spectrometry.
The method for determining the data of the ultraviolet absorption spectrum, the fluorescence spectrum and the fluorescence spectrum among the systems by a molar ratio method comprises the following steps: g and Q [8} were prepared as 1.0mmol/L and 0.1mmol/L aqueous solutions (pH 4) respectively, with a guest concentration of 0.04mmol/L and Q [8] was changed]Concentration of (2) configuration NQ[8]/NGAn aqueous solution (pH 4) of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, the uv-visible absorption spectrum of the solution was measured at room temperature (experimental data in fig. 1(a), fig. 1 (B)); the concentration of the immobilized guest is 0.02mmol/L, and Q8 is changed]Concentration of (2) configuration NQ[8]/NGMeasuring fluorescence emission spectrum of the solution at excitation wavelength of 346nm, excitation slit of 5nm, emission slit of 5nm and voltage of 540V for 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.6, 1.2, 1.4, 1.6, 2.0, 2.2, 2.4, 2.6, 2.8 and 3.0 (experimental data is shown in figure 2(A) and figure 2(B)). then measuring ultraviolet absorption spectrum and fluorescence spectrum between systems by using equimolar continuous variation method (JOB method), fixing total concentration of subject and object to be 4.0mmol/L, and preparing a series of different molar ratios N by continuously varying the ratio of the amount of substance between the subject and objectQ[8]/(NQ[8]+NG) 0.1, 0.2. cndot. cndot.0.8, 0.9, and according to this method, the ultraviolet absorption spectrum and the fluorescence spectrum were measured (experimental data are shown in FIG. 1(C) and FIG. 2 (C)).
Experimental example 2 quantitative analysis
The concentration obtained by the invention is 2.0 x 10-5The detection results are shown in FIG. 6, and it can be seen that the concentrations of L-Phe in the standard solutions are different after different volume fractions are added, the different concentrations of L-Phe can cause the fluorescent probe solutions to generate different degrees of fluorescence sensitization, and the linear range of the L-Phe response is (1.0-30.0) < 10 > L-Phe response-5mol/L, detection limit of 2.108 x 10-6mol/L. Then, a nuclear magnetic titration experiment was conducted in which a probe standard solution was added to a solution containing L-Phe at a different concentration (the results are shown in FIG. 8), and it was concluded that the mode of action of L-Phe with the probe is shown in FIG. 8.
The concentration obtained by the invention is 2.0 x 10-5The test results are shown in FIG. 7, and it can be seen that the concentrations of L-Trp in the standard solutions are different after different volume fractions are added, the different concentrations of L-Trp can cause the fluorescence sensitization of the fluorescent probe solutions to different degrees, and the linear range of the L-Trp response is (1.0-30.0) < 10 >-5mol/L, detection limit 4.750 x 10-6mol/L. Then, a nuclear magnetic titration experiment was conducted in which a probe standard solution was added to a solution containing L-Phe at a different concentration (the result of the detection is shown in FIG. 9), and it was deduced from this that the mode of action of L-Trp with the probe is shown in FIG. 9.
Compared with the prior art, the invention has the following beneficial effects:
1. the fluorescent probe can simultaneously detect basic L-amino acids (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in water, can detect only one or more amino acids, and has the advantages of wide detection range and specificity. The method has the advantages that one probe is used for detecting various amino acids, the detection of various target amino acids by a single probe can be realized, the detection cost is low, the detection efficiency is high, and the analysis of a complex microscopic system is facilitated.
2. The invention uses Q8 and anti-acridine hydrochloride to form supermolecule complex, when the above amino acid is detected, basic L-amino acid (L-arginine, L-histidine, L-lysine) can destroy the acid-base environment of probe to make the fluorescence of probe generate blue shift, phenylalanine and tryptophan destroy probe to form new complex to make the fluorescence of probe generate sensitization action, so that it can simply, quickly and sensitively detect basic L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in water.
3. Compared with the traditional detection method, the detection cost is greatly reduced, the operation is convenient, and the real-time detection can be realized.
Therefore, the probe can detect basic L-amino acids (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in water, one probe can detect a plurality of amino acids, the detection of a single probe on a plurality of target amino acids can be realized, the detection cost is low, the detection efficiency is high, and the analysis of a complex microscopic system is facilitated. In addition, when the probe is used for detection, the probe also has the characteristics of higher sensitivity, simple sample treatment, convenient operation, quick determination and real-time detection.
Description of the drawings:
FIG. 1 shows the UV light of Q8 in mole ratio with acridine hydrochloride (A), (B), JOB (C);
FIG. 2 shows fluorescence of Q8 in the molar ratio of acridine hydrochloride (A), (B) and JOB (C);
FIG. 3 is a diagram of NMR titration and inclusion patterns of Q8 and acridine hydrochloride; wherein, (A) G; (B) g, Q [8] ═ 1: 0.17; (C) g, Q [8] ═ 1: 0.42; (D) g, Q [8] ═ 1: 0.69; (E) g, Q [8] ═ 1: 1.04; (F) q < 8 >;
FIG. 4 is a mass spectrum of a probe formed by the inclusion of Q8 with acridine hydrochloride;
FIG. 5 is a graph showing fluorescence spectra when a probe standard solution is added to solutions containing different L-amino acids;
FIG. 6 is a graph showing a simulation of fluorescence spectrum curve (A), fluorescence intensity (B) and detection limit (C) when a probe standard solution is added to solutions containing L-Phe at different concentrations;
FIG. 7 is a graph showing the fluorescence spectrum curve (A), fluorescence intensity (B) and detection limit simulation (C) of the probe standard solution added with solutions containing L-Trp at different concentrations;
FIG. 8 is a graph showing nuclear magnetic titration of a standard solution of a probe added to solutions containing L-Phe at different concentrations; wherein, (A) G; (B) g, Q [8] ═ 1: 1; (C) q [8] L-Phe ═ 1: 1: 7.15 of; (D) q [8] L-Phe ═ 1: 1: 14.33; (E) q [8] L-Phe ═ 1: 1: 21.26; (F) q [8] L-Phe ═ 1: 1: 27.78; (G) L-Phe;
FIG. 9 is a nuclear magnetic titration chart of a probe standard solution added to solutions containing L-Trp at different concentrations; wherein, (A) G; (B) g, Q [8] ═ 1: 1; (C) q [8] L-Trp is 1: 1: 0.7; (D) q [8] L-Trp is 1: 1: 1.75(E) G: Q [8] L-Trp ═ 1: 1: 3.97 of the total weight of the mixture; (F) q [8] L-Trp is 1: 1: 5.88; (G) q [8] L-Trp is 1: 1: 7.63; (H) q [8] L-Trp is 1: 1: 10.68; (I) q [8] L-Trp is 1: 1: 17.01; (D) L-Trp.
Detailed Description
Example 1:
a fluorescent probe for detecting multiple amino acids in water, the molecular formula of said probe is C48H48N32O16@C13H10NCl, the probe pattern of the probe is shown in figure 3, the probe is composed of Q [8]]And acridine hydrochloride according to a molar ratio of 1:1, preparing the composition.
The preparation method of the fluorescent probe for detecting various L-amino acids in water comprises the following steps:
1) taking Q [8]]Mixing Q [8]]Adding secondary water with pH 7 to dissolve to obtain solution A with concentration of 1.0 × 10-4mol/L；
2) Dissolving acridine hydrochloride with secondary water having pH of 7 to obtain solution B with concentration of 1.0 × 10-3mol/L；
3) Mixing solution A and solution B according to the weight ratio of 10: 1 (i.e. molar ratio 1: 1), the result is obtained by the ultraviolet and fluorescence molar ratio of Q8 and acridine hydrochloride, JOB experiment, mass spectrum experiment and nuclear magnetic titration of Q8 and acridine hydrochloride (the experimental results are shown in figure 1, figure 2, figure 3 and figure 4), the reaction is carried out at normal temperature, and the pH is adjusted to 4 by concentrated hydrochloric acid to obtain the probe.
The method for detecting the basic L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in the water by the fluorescent probe comprises the following steps:
1) diluting the probe with pH 4 water to obtain a concentration of 2.0 x 10-5A probe standard solution of mol/L;
2) adding water to be detected into the probe standard solution prepared in the step 1), standing for 15min, then performing fluorescence emission spectrometry at a fixed excitation wavelength of 346nm, and drawing a change curve of fluorescence intensity at the excited laser wavelength;
3) calculating the change value delta F of the fluorescence emission spectrum intensity corresponding to 478nm before and after the water to be detected is added into the fluorescent probe solution according to the curve of the step 2), and indicating that the water to be detected contains basic L-amino acid (L-arginine, L-histidine and L-lysine) when the fluorescence emission spectrum intensity corresponding to 478nm before and after the water to be detected is added generates obvious blue shift; when the fluorescence emission spectrum intensity corresponding to 478nm before and after the water to be detected is added is obviously enhanced, the water to be detected contains L-phenylalanine and L-tryptophan. When the fluorescence emission spectrum intensity at 478nm does not occur before and after the water to be detected is added, the fluorescence emission spectrum intensity indicates that the water to be detected does not contain basic L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan. The results are shown in FIG. 5.
Example 2:
a fluorescent probe for detecting multiple amino acids in water is prepared from Q8 and acridine hydrochloride in the molar ratio of 1: 0.5.
The preparation method of the fluorescent probe for detecting various L-amino acids in water comprises the following steps:
1) taking Q [8]]Mixing Q [8]]Adding secondary water with pH 7 to dissolve to obtain solution A with concentration of 1.0 × 10-4mol/L；
2) Dissolving acridine hydrochloride with secondary water having pH of 7 to obtain solution B with concentration of 1.0 × 10-3mol/L；
3) Mixing solution A and solution B according to the weight ratio of 10: 1 (i.e. molar ratio 1: 1), the result is obtained by the ultraviolet and fluorescence molar ratio of Q8 and acridine hydrochloride, JOB experiment, mass spectrum experiment and nuclear magnetic titration of Q8 and acridine hydrochloride (the experimental results are shown in figure 1, figure 2, figure 3 and figure 4), the reaction is carried out at normal temperature, and the pH is adjusted to 4 by concentrated hydrochloric acid to obtain the probe.
The method for detecting the basic L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in the water by the fluorescent probe comprises the following steps:
1) diluting the probe with pH 4 water to obtain a concentration of 2.0 x 10-5A probe standard solution of mol/L;
2) adding water to be detected into the probe standard solution prepared in the step 1), standing for 15min, then performing fluorescence emission spectrometry at a fixed excitation wavelength of 346nm, and drawing a change curve of fluorescence intensity at the excited laser wavelength;
3) calculating the change value delta F of the fluorescence emission spectrum intensity corresponding to 478nm before and after the water to be detected is added into the fluorescent probe solution according to the curve of the step 2), and indicating that the water to be detected contains basic L-amino acid (L-arginine, L-histidine and L-lysine) when the fluorescence emission spectrum intensity corresponding to 478nm before and after the water to be detected is added generates obvious blue shift; when the fluorescence emission spectrum intensity corresponding to 478nm before and after adding the water to be detected is obviously enhanced, the water to be detected contains phenylalanine and tryptophan. When the fluorescence emission spectrum intensity corresponding to 478nm does not occur before and after the water to be detected is added, the water to be detected does not contain alkaline L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan.
Example 3:
a fluorescent probe for detecting multiple metal ions in water is prepared from Q8 and acridine hydrochloride in the molar ratio of 1: 2, and (3) preparing.
The preparation method of the fluorescent probe for detecting various L-amino acids in water comprises the following steps:
1) taking Q [8]]Mixing Q [8]]Adding secondary water with pH 7 to dissolve to obtain solution A with concentration of 1.0 × 10-4mol/L；
2) Acridine hydrochloride, acridine hydrochloride pHDissolving with secondary water of 7 to obtain solution B with concentration of 1.0 x 10-3mol/L；
3) Mixing solution A and solution B according to the weight ratio of 10: 1 (i.e. molar ratio 1: 1), the result is obtained by the ultraviolet and fluorescence molar ratio of Q8 and acridine hydrochloride, JOB experiment, mass spectrum experiment and nuclear magnetic titration of Q8 and acridine hydrochloride (the experimental results are shown in figure 1, figure 2, figure 3 and figure 4), the reaction is carried out at normal temperature, and the pH is adjusted to 4 by concentrated hydrochloric acid to obtain the probe.
The method for detecting the basic L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan in the water by the fluorescent probe comprises the following steps:
1) diluting the probe with pH 4 water to obtain a concentration of 2.0 x 10-5A probe standard solution of mol/L;
2) adding water to be detected into the probe standard solution prepared in the step 1), standing for 15min, then performing fluorescence emission spectrometry at a fixed excitation wavelength of 346nm, and drawing a change curve of fluorescence intensity at the excited laser wavelength;
3) calculating the change value delta F of the fluorescence emission spectrum intensity corresponding to 478nm before and after the water to be detected is added into the fluorescent probe solution according to the curve of the step 2), and indicating that the water to be detected contains basic L-amino acid (L-arginine, L-histidine and L-lysine) when the fluorescence emission spectrum intensity corresponding to 478nm before and after the water to be detected is added generates obvious blue shift; when the fluorescence emission spectrum intensity corresponding to 478nm before and after the water to be detected is added is obviously enhanced, the water to be detected contains L-phenylalanine and L-tryptophan. When the fluorescence emission spectrum intensity corresponding to 478nm does not occur before and after the water to be detected is added, the water to be detected does not contain alkaline L-amino acid (L-arginine, L-histidine, L-lysine), L-phenylalanine and L-tryptophan.
Claims (4)
1. A fluorescent probe, characterized in that: the molecular formula of the fluorescent probe is C48H48N32O16@C13H10NCl; the preparation method of the fluorescent probe comprises the following steps: is prepared from eight-membered cucurbituril (Q8)]) With acridine saltsPreparing acid salt; said Q [8]The molar ratio of the compound to the acridine hydrochloride is 1: 1; the method specifically comprises the following steps:
(1) taking Q [8]]，Q[8]Dissolving in water to obtain solution A with concentration of 1.0 x 10-4mol/L；
(2) Dissolving acridine hydrochloride in water to obtain solution B with concentration of 1.0 × 10-3mol/L；
(3) Mixing solution A and solution B according to the weight ratio of 10: 1, namely mixing in a molar ratio of 1:1, reacting at normal temperature, and adjusting the pH value to 4 to obtain the fluorescent probe.
2. Use of a fluorescent probe according to claim 1, characterized in that: used for detecting alkaline L-amino acid, L-phenylalanine and L-tryptophan in water; the basic L-amino acids are L-arginine, L-histidine and L-lysine.
3. Use of a fluorescent probe according to claim 2, characterized in that: when the method is used for detecting alkaline L-amino acid, L-phenylalanine and L-tryptophan in water, the specific detection method comprises the following steps:
1) diluting the fluorescent probe with pH 4 water to 2.0 x 10-5Obtaining a probe standard solution by using a mol/L solution;
2) adding water to be detected into the probe standard solution prepared in the step 1), standing for 10-20min, then performing fluorescence emission spectrometry at a fixed excitation wavelength of 346nm, and drawing a change curve of the fluorescence intensity at the excited laser wavelength;
3) calculating a fluorescence emission spectrum intensity change value delta F corresponding to 478nm before and after the water to be detected is added into the fluorescent probe solution according to the curve of the step 2), namely detecting basic L-amino acid, L-phenylalanine and L-tryptophan in the water, wherein the basic L-amino acid is L-arginine, L-histidine or L-lysine.
4. Use of a fluorescent probe according to claim 3, characterized in that: in the step 3), when the fluorescence emission spectrum intensity corresponding to 478nm changes or blue shifts occur before and after the water to be detected is added, the water to be detected contains basic L-amino acid, L-phenylalanine and L-tryptophan, wherein the basic L-amino acid is L-arginine, L-histidine or L-lysine; when the fluorescence emission spectrum intensity of the sample before and after the sample is added and under the condition of 478nm does not change or blue shift, the sample is indicated to contain no basic L-amino acid, L-phenylalanine and L-tryptophan, wherein the basic L-amino acid is L-arginine, L-histidine or L-lysine.
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| CN110412000B (en) * | 2019-07-31 | 2022-05-17 | 贵州大学 | Fluorescent probe for detecting L-tryptophan based on ten-element cucurbituril and detection method thereof |
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| CN112362628A (en) * | 2020-11-12 | 2021-02-12 | 贵州师范学院 | Fluorescent probe for detecting L-tryptophan |
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| CN115340615B (en) * | 2022-08-12 | 2023-05-02 | 同济大学 | Fluorescent molecule based on cyclodextrin-amino acid and synthetic method and application thereof |
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