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CN-122010940-A - Citric acid-based fluorescent dye with long-life characteristic of water, and preparation method and application thereof

CN122010940ACN 122010940 ACN122010940 ACN 122010940ACN-122010940-A

Abstract

The invention relates to the technical field of fluorescence imaging, and provides a citric acid-based fluorescent dye with long-life water-induced property, and a preparation method and application thereof. The provided citrate-based fluorescent dye has obvious characteristic of 'long life due to water' in physiological water environment, has the service life of 15 ns, can perform FLIM analysis in a long-life window, effectively reduces the interference of short-life endogenous background fluorescence, and obviously improves the contrast and quantitative accuracy of imaging.

Inventors

  • YANG JIAN
  • WANG RONGCHEN
  • Ding Linyu

Assignees

  • 西湖大学

Dates

Publication Date
20260512
Application Date
20260409

Claims (10)

  1. 1. A citric acid-based fluorescent dye with long lifetime characteristics due to water, characterized in that the structural formula of the dye is shown as follows: TPA-ER。
  2. 2. a method for preparing a citrate-based fluorescent dye having long life-span characteristics as recited in claim 1, comprising the steps of: Adding potassium carbonate into the compound TPA-N-COOH for stirring reaction, and then adding N- (2-bromoethyl) -4-methylbenzenesulfonamide for heating reaction to obtain a compound TPA-ER; The synthetic route is as follows: 。
  3. 3. the method for producing a citric acid-based fluorescent dye having a long life property due to water as claimed in claim 2, wherein, The reaction condition of the compound TPA-N-COOH and potassium carbonate is that stirring reaction is carried out at 30-40 ℃ for 20-30 min.
  4. 4. A process for preparing a citrate fluorescent dye having long lifetime characteristics due to water as claimed in claim 2 or 3, The heating reaction condition is that stirring reaction is carried out for 18-24 hours at the temperature of 30-40 ℃.
  5. 5. A process for preparing a citrate fluorescent dye having long lifetime characteristics due to water as claimed in claim 2 or 3, The mol ratio of TPA-N-COOH to potassium carbonate to N- (2-bromoethyl) -4-methylbenzenesulfonamide is 1:2:1.5.
  6. 6. A process for preparing a citrate fluorescent dye having long lifetime characteristics due to water as claimed in claim 2 or 3, And after the heating reaction is finished, washing, extracting, drying and/or purifying steps are further included, wherein in the purifying step, column chromatography is adopted for separation and purification, and the chromatographic liquid is petroleum ether and ethyl acetate=1:1, and V/V.
  7. 7. A process for preparing a citrate fluorescent dye having long lifetime characteristics due to water as claimed in claim 2 or 3, The method also comprises the steps of heating citric acid and 1, 2-diphenyl ethylenediamine to react and obtain a compound TPA-N-COOH; 。
  8. 8. the method for producing a citric acid-based fluorescent dye having a long life property due to water as claimed in claim 7, wherein, The heating reaction condition is that stirring reaction is carried out at 120-140 ℃ for 4-8 h; and/or the molar ratio of the citric acid to the 1, 2-diphenyl ethylenediamine is 1:1; and/or after the heating reaction is finished, washing, extracting, drying and/or purifying steps are further included, wherein in the purifying step, column chromatography is adopted for separation and purification, and the chromatographic liquid is dichloromethane, methanol=8:1, and V/V.
  9. 9. Use of the citrate-based fluorescent dye with long-life property due to water according to claim 1 or the compound prepared by the preparation method of the citrate-based fluorescent dye with long-life property due to water according to any one of claims 2 to 8 in the field of fluorescence imaging.
  10. 10. The use according to claim 9, characterized in that it comprises: (1) Use in the field of fluorescence lifetime imaging; (2) Monitoring the hydration state of the microenvironment in the cell; (3) The dynamic change of the hydrophobic microenvironment accompanying early stage of the cytoplasmic network stress is monitored.

Description

Citric acid-based fluorescent dye with long-life characteristic of water, and preparation method and application thereof Technical Field The invention relates to the technical field of fluorescence imaging, in particular to a citric acid-based fluorescent dye with a water-induced long-life characteristic, and a preparation method and application thereof. Background Fluorescent Lifetime Imaging (FLIM) achieves quantitative analysis by detecting the average residence time of fluorescent molecules in an excited state, and because signals are not easily affected by probe concentration, excitation light intensity or biological tissue light scattering, higher accuracy and reliability than intensity imaging are exhibited in life analysis applications. However, the performance of FLIM technology is largely dependent on the development of high performance fluorescent probes. At present, commonly used long-life probes (such as rare earth complexes) often face the problems of poor biocompatibility, difficult regulation and control of optical properties and the like. Compared with the organic micromolecular dye, the organic micromolecular dye has the characteristics of excellent biocompatibility and easiness in modification, but the fluorescent lifetime is generally short (usually 0-5 ns), and the organic micromolecular dye is seriously overlapped with the autofluorescence lifetime (0-7 ns) of cells, so that the signal-to-back ratio is low and the detection sensitivity is limited. On the other hand, probes constructed based on such dyes typically have a small lifetime response to changes in the intracellular microenvironment (often below 1 ns), which limits to some extent the contrast and quantification accuracy of FLIM imaging. Particularly, water is used as a main component of a living system, and the fluorescence life of most organic dyes is further shortened by enhancing non-radiative transition ways such as molecular vibration, collision quenching and the like, so that the imaging contrast and the quantitative capability of FLIM in a physiological environment are severely restricted. Thus, the development of high performance fluorescent probes that are capable of maintaining long life in aqueous environments and have a significant response to changes in cellular microenvironments has become a key challenge in advancing FLIM technology for accurate biological imaging. Disclosure of Invention The invention aims to overcome the defects in the prior art and provides a citric acid-based fluorescent dye with long fluorescence lifetime, and a preparation method and application thereof. The citric acid-based fluorescent dye provided by the invention is characterized by exhibiting unique characteristic of long life due to water. In particular, the fluorescence lifetime of the dye in an aqueous environment is significantly prolonged compared to that in an organic solvent, up to 19.5 ns, enabling high signal-to-noise fluorescence lifetime imaging in long-lived intervals (e.g., greater than 10 nanoseconds). The fluorescence lifetime of the dye is highly sensitive to changes in ambient water content, and changes in the hydration state of the microenvironment can be effectively reflected by quantitative changes in lifetime. In addition, the dye also has endoplasmic reticulum targeting capability and can be specifically enriched in the endoplasmic reticulum of living cells. Based on the characteristics, the fluorescent dye is particularly suitable for fluorescent lifetime imaging in living cells, can be used for monitoring the dynamic change of the hydrophobic microenvironment accompanying early stage of endoplasmic reticulum stress, and provides a novel and effective probe tool for real-time research and quantitative analysis of related pathophysiological processes. The invention also provides a preparation method of the fluorescent dye, which has the advantages of easily available raw materials, mild reaction conditions, simple steps and easy implementation. The technical scheme adopted by the invention is as follows: The embodiment of the invention provides a citric acid-based fluorescent dye with long fluorescence lifetime in a physiological aqueous phase environment, which has the following structure: TPA-ER。 The embodiment of the invention provides a preparation method of the compound TPA-ER, which comprises the following steps: Adding potassium carbonate into the compound TPA-N-COOH for stirring reaction, and then adding N- (2-bromoethyl) -4-methylbenzenesulfonamide for heating reaction to obtain a compound TPA-ER; The synthetic route is as follows: 。 In some embodiments, the reaction conditions of the compound TPA-N-COOH and potassium carbonate are from 30 to 40 ℃ and stirring reaction is from 20 to 30 min, e.g., the heating temperature can be any one or range between any two of 30, 35, 40 ℃. For example, the heating time may be any one of 20, 25, 30 min or a range between any two values. In some embodiments, the heating