CN114213388B - Use of fluorescent probes based on thiophene compounds for detecting polarity and viscosity values - Google Patents

Use of fluorescent probes based on thiophene compounds for detecting polarity and viscosity values Download PDF

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CN114213388B
CN114213388B CN202111257460.9A CN202111257460A CN114213388B CN 114213388 B CN114213388 B CN 114213388B CN 202111257460 A CN202111257460 A CN 202111257460A CN 114213388 B CN114213388 B CN 114213388B
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polarity
viscosity
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CN114213388A (en
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谢振达
赖燕琴
满菊
姜春筱
周弈宇
李娜
杨仲毅
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Taizhou University
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Abstract

The application discloses application of a thiophene compound-based fluorescent probe in detecting a polarity value and a viscosity value, and the inventor of the application finds that the thiophene compound with the structural formula shown in the formula (1) can detect the polarity value under the condition that 399nm is an excitation wavelength and 485nm is an emission wavelength fluorescent channel in experiments; the viscosity value can be detected under the fluorescent channel with the excitation wavelength of 440nm and the emission wavelength of 595 nm. In addition, the probe has good anti-interference capability and low cytotoxicity, can realize the respective detection of the polarity value and the viscosity value, and has potential application value in the field of biological detection.

Description

Use of fluorescent probes based on thiophene compounds for detecting polarity and viscosity values
Technical Field
The application relates to the technical field of fluorescent probe detection, in particular to a new application of a known compound in detecting a polarity value and a viscosity value of a solution.
Background
Fluorescent probes have been used to detect the concentration or activity of small active molecules, enzymes, metal ions in living organisms, and have revealed mechanisms of occurrence of various diseases and metabolic pathways of analytes, due to the advantages of high throughput detection, high selectivity, high sensitivity, small damage to cell/living sample, etc. Currently, for the field of fluorescent probes, the future probe development trend is as follows: 1) The preparation method is economical and is more suitable for industrial production; 2) The detection object is multifunctional, and the research on the interrelation of multiple analytes under the same disease model can be realized by using a single probe.
Many studies have found that the occurrence of the disease is closely related to changes in the intracellular environment. For example, the intracellular environment of a tumor has a higher viscosity value and a lower polarity value than that of a normal cell. Currently, most probes can only detect viscosity or polarity values in cells singly, which is not conducive to multi-factor analysis of disease models. Therefore, the preparation and discovery of the bifunctional probe capable of realizing the detection of the viscosity value and the polarity value have important research value and application value. In view of the above, the present invention is specifically proposed.
Disclosure of Invention
The application provides a new application of a compound in qualitative detection of a polarity value and a viscosity value of a solution.
The structural formula of the compound is shown as the formula (1):
Figure BDA0003324554060000021
this compound is a known compound, and its preparation method can be found in (Li S, wang P, feng W, et al. Organic imaging of mitochondrial vision and hydrogen peroxide in Alzheimer' S disease by a single road-in free fluorescent probe with a large storage beds shift [ J ]. Chemical Communications,2020,56, 1050-1053).
The compound is a traditional push-pull fluorophore, making possible the ability of the probe to respond to polar values; on the other hand, the compound has a freely rotatable aldehyde group and an N, N-dimethyl group, so that the ability of the compound to respond to viscosity values is made possible. The inventor of the application finds that the compound has double response functions of a polarity value and a viscosity value in a fluorescence detection experiment by using a fluorescence spectrophotometer daily.
Based on the application, the application provides the application of the compound with the structural formula shown as the formula (1) in detecting the polarity value and the viscosity value.
The invention also provides application of the compound with the structural formula shown in the formula (1) in preparation of products for detecting the polarity value and the viscosity value.
Optionally, the product is a reagent or a kit
Optionally, the polarity and viscosity values are those of a solution or within a cell.
The application also provides a kit for detecting the polarity value and the viscosity value of a solution or a cell, which comprises a compound with a structural formula shown in a formula (1):
Figure BDA0003324554060000022
the application also provides a method for detecting a polarity value and a viscosity value in a solution, which comprises the following steps:
adding a compound with a structural formula shown in a formula (1) into a solution to be detected for reaction, measuring the polarity change of the solution to be detected in a reaction liquid obtained after the reaction is finished under the conditions that the excitation wavelength is 399nm and the emission wavelength is 485nm, and measuring the viscosity change of the solution to be detected under the conditions that the excitation wavelength is 440nm and the emission wavelength is 595 nm.
Optionally, when detecting a polarity change, a 1,4-dioxane-water mixing system is selected as a polarity test system, the viscosity values of 1,4-dioxane and water are almost equal, the polarity value of 1,4-dioxane is small, and the polarity value of water is large.
Optionally, when the viscosity change is detected, a glycerol-ethanol mixture system is selected as a viscosity test system, the polarity values of glycerol and ethanol are almost equal, the viscosity value of glycerol is large, and the polarity value of ethanol is small.
The detection method for qualitatively detecting the polarity change in the solution comprises the following steps:
adding the difunctional fluorescent probe into 1,4-dioxane-water mixed solution, uniformly mixing, collecting the fluorescence intensity of the solution to be detected under the conditions that the excitation wavelength is 399nm and the emission wavelength is 485nm, and comparing the fluorescence intensity under different 1,4-dioxane-water ratios.
And qualitatively detecting the viscosity change in the solution, wherein the detection method comprises the following steps:
and adding the difunctional fluorescent probe into the glycerol-ethanol mixed solution, uniformly mixing, collecting the fluorescence intensity of the solution to be detected under the conditions that the excitation wavelength is 440nm and the emission wavelength is 595nm, and comparing the fluorescence intensities under different glycerol-ethanol ratios.
The application has at least the following beneficial effects:
the difunctional fluorescent probe (I) can realize the respective detection of the polarity value and the viscosity value of the solution under different fluorescent channels, improves the utilization rate of the probe and the visualization performance of cells/living bodies, and has good scientific research and practical application values.
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FIGS. 1 to 2 show nuclear magnetic hydrogen spectra and carbon spectra of the fluorescent probe (I).
FIG. 3 is a graph showing fluorescence intensities of the mixed system of the fluorescent probe (I) at different ratios of 1,4-dioxane-water at an excitation wavelength of 399nm and an emission wavelength of 485 nm.
FIG. 4 is a fluorescence spectrum of the fluorescent probe (I) in a mixed system at different glycerol-ethanol ratios, with an excitation wavelength of 440nm and an emission wavelength of 595 nm.
FIG. 5 is a graph of the interference-free fluorescence intensity of the fluorescent probe (I).
FIG. 6 is a cytotoxicity test chart of the fluorescent probe (I).
Detailed Description
The technical solutions of the present application will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without any creative effort belong to the protection scope of the present application.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the present application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Example 1: preparation of bifunctional fluorescent Probe (I)
Under nitrogen atmosphere, compound 1,4-bromo-N, N-dimethylaniline (5 mmol) and catalyst Pd (PPh) 3 ) 4 (0.43 mmol) was taken up in 15mL of tetrahydrofuran, followed by the addition of K 2 CO 3 Aqueous solution (2M, 1.25 mL). After 1 hour, 5-formyl-2-thiopheneboronic acid (10 mmol) dissolved in 10mL of tetrahydrofuran was slowly added to the reaction solution and the reaction was stirred at 80 ℃ for 12 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NaCl solution, extracted three times with dichloromethane, and the organic layer was dried with anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with methylene chloride/petroleum ether (v/v, 1:1) to give fluorescent probe (I) (0.29 g,yield 25.2%).
The synthetic route is as follows:
Figure BDA0003324554060000051
the nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum of the fluorescent probe (I) are shown in FIG. 1 and FIG. 2, respectively. 1 H NMR(400 MHz,Chloroform-d)δ9.82(s,1H),7.68(d,J＝4.0Hz,1H),7.56(d,J＝9.0Hz,2H), 7.24(d,J＝4.1Hz,1H),6.72(d,J＝8.4Hz,2H),3.02(s,6H). 13 C NMR(101MHz, Chloroform-d)δ182.43,156.01,151.09,140.10,138.07,127.48,121.51,112.17, 40.24.
Example 2: fluorescence spectroscopy of bifunctional fluorescent probes (I) (10. Mu.M) for polar response.
Accurately weighing a certain amount of the difunctional fluorescent probe (I), preparing a mother solution with the concentration of 10mM by using dimethyl sulfoxide, sucking 4 mu L of the difunctional fluorescent probe by a liquid transfer gun, adding the difunctional fluorescent probe into 4mL of mixed solvent in different 1,4-dioxane-water ratios, shaking, uniformly mixing, adding the mixture into a cuvette, and measuring the fluorescence spectra of the probe (I) in different mixed systems by using a fluorescence spectrophotometer.
As can be seen from FIG. 3, as the proportion of 1,4-dioxane in the mixed solution decreases, the polarity of the solution increases, and the fluorescence intensity with 399nm as the excitation wavelength and 485nm as the emission wavelength decreases, demonstrating that the probe has the response capability to the polarity of the solution.
Example 3: fluorescence spectroscopy of bifunctional fluorescent probes (I) (10. Mu.M) for viscosity response.
Accurately weighing a certain amount of the difunctional fluorescent probe (I), preparing a mother solution with the concentration of 10mM by using dimethyl sulfoxide, sucking 4 mu L of the difunctional fluorescent probe by a liquid transfer gun, adding the difunctional fluorescent probe into 4mL of mixed solvent with different glycerol-ethanol ratios, shaking, uniformly mixing, adding the mixture into a cuvette, and measuring the fluorescence spectra of the probe (I) in different mixed systems by using a fluorescence spectrophotometer.
As can be seen from FIG. 4, as the ratio of glycerol in the mixture solution increased, the viscosity of the solution increased, and the fluorescence intensity increased with 440nm as the excitation wavelength and 595nm as the emission wavelength, demonstrating that the probe had a response capability to the viscosity of the solution.
Example 4: and (3) testing the anti-interference capability of the bifunctional fluorescent probe (I).
A certain amount of the fluorescent probe (I) is accurately weighed and prepared into a 10mM stock solution by using dimethyl sulfoxide, and on one hand, a pipette gun sucks 4 mu L of the stock solution and adds the stock solution into 4mL of PBS solution containing different analytes (1 to 11 are respectively Na) 2 SO 4 、NaNO 2 、Na 2 SO 3 、NaHSO 3 、CH 3 COONa、Na 2 CO 3 NaF, KBr, KI, naClO, 1,4-Dioxane), shaking, uniformly mixing, adding into a cuvette after ultrasonic treatment, and testing by using a fluorescence spectrophotometer with 399nm as an excitation wavelength and 485nm as an emission wavelength; on the other hand, the pipette is pipetted 4. Mu.L into 4mL of PBS containing different analytes (Na for 1 to 17, respectively) 2 SO 4 、NaNO 2 、Na 2 SO 3 、NaHSO 3 、CH 3 COONa、Na 2 CO 3 、NaF、KBr、KI、 Cys、GSH、Hcy、NaHS、H 2 O 2 NaClO, PBS and glycerol), shaking, mixing uniformly, adding into a cuvette after ultrasonic treatment, and testing by using a fluorescence spectrophotometer with 440nm as an excitation wavelength and 595nm as an emission wavelength;
as shown in FIG. 5a, the addition of other analytes had little effect on the fluorescence intensity of the probe at 485nm compared to 1,4-dioxane; as shown in FIG. 5b, the addition of the other analyte had little effect on the fluorescence intensity of the probe at 595nm compared to glycerol. The experimental results prove that the probe has good specificity on both the polarity value and the viscosity value.
Example 5: and (3) toxicity test of the fluorescent probe (I) to Hela cells.
The MTT cytotoxicity experiment is utilized to test the toxicity of the fluorescent probe (I) to Hela cells. After Hela cells were incubated with the probe culture medium containing different concentrations for 24h, the survival rate of the cells was calculated.
As shown in FIG. 6, the cell viability reached as high as 80% or more even at a probe concentration of 20. Mu.M, demonstrating that the probe is less toxic to the cells and has potential for use in cell imaging.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent application shall be subject to the appended claims.

Claims (4)

1. The application of the compound with the structural formula shown as the formula (I) in preparing products for detecting polarity values and viscosity values,
Figure FDA0004001566100000011
2. use according to claim 1, wherein the polarity and viscosity values are those in solution or in cells.
3. Use according to claim 1, wherein the product is a reagent or kit.
4. A kit for detecting the polarity value and the viscosity value of a solution or a cell, characterized in that it comprises a compound of formula (I):
Figure FDA0004001566100000012
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