Waveform protein response type wash-free fluorescent probe as well as preparation method and application thereof

A technology of vimentin and fluorescent probes, which is applied in the field of vimentin-responsive wash-free fluorescent probes and its preparation, can solve problems such as unsatisfactory specificity and accuracy, reduced detection sensitivity, and detection sample interference, and achieve high Sensitivity, low cost, guaranteed effect of charge interaction

Active Publication Date: 2019-12-17
THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, conventional fluorescent probes often produce fluorescent background due to non-specific binding or excess probes, resulting in reduced detection sensitivity or false positive results, so stained samples need to be washed multiple times, which will also affect the detection Unexpected interference and damage to the sample
There is no specific, accurate, fast, and simple assay method in the prior art, and there are certain defects, which cannot meet the requirements of specificity and accuracy.

Method used

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  • Waveform protein response type wash-free fluorescent probe as well as preparation method and application thereof
  • Waveform protein response type wash-free fluorescent probe as well as preparation method and application thereof
  • Waveform protein response type wash-free fluorescent probe as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0040] Such as figure 1 , Provide vimentin-specific responsive fluorescent probe vim-TPE-1 / 2, including aggregation-inducing luminescent fluorescent groups and peptides with affinity for vimentin, as well as the connecting group between the two.

[0041] The aggregation-induced luminescence fluorescent group is 1,2-diphenyl-1,2-bis(4-aminophenyl)ethylene.

[0042] The amino acid peptide fragment with affinity for vimentin contains the following sequence SEQ ID NO:1: CVNTANST.

[0043] The linking group between the aggregation-induced luminescent fluorescent group and the affinity peptide for vimentin is 6-maleimidohexanoic acid.

[0044] In a vimentin-specific responsive fluorescent probe molecule, the number of vimentin affinity peptides and linking groups is two each, and the relative position of the peptides is the cis or trans of the tetrastyrene double bond.

Embodiment 2

[0046] Synthesis of Vim-TPE-1 / 2 Specific Response Fluorescent Probe

[0047] See the following reaction formula (1):

[0048] (1)

[0049] Dissolve 1,2-diphenyl-1,2-bis(4-aminophenyl)ethylene (cis-trans isomer mixture, 200 mg, 1 equiv) in 10 ml dichloromethane, 6-maleimide Aminocaproic acid (350 mg, 3 equiv) and EEDQ (408 mg, 3 equiv) were added to the reaction solution to carry out the amide condensation reaction of the carboxyl group and the primary amine, and react at room temperature overnight. The solvent was spin-dried with a rotary evaporator, and petroleum ether: ethyl acetate 1:1 was separated by silica gel column to obtain two cis-trans isomer intermediates cis-TPE-1 156 mg and trans-TPE-2 25 mg, such as Figure 2-6 , By 1H NMR ( figure 2 ), 13C NMR ( image 3 ) COSY ( Figure 4 with Figure 5 ), NOESY ( Image 6 ) Two-dimensional nuclear magnetic resonance spectroscopy to determine the structure.

[0050] cis-TPE-1: 1H NMR (400 MHz, Pyridine-d5) δ 10.57 (s, 2H), 7.81 (d,...

Embodiment 3

[0063] Imaging detection of vim-TPE-1 and vim-TPE-2 on human lung adenocarcinoma cells NCI-H1975 and PC-9 cells

[0064] Human lung cancer cells NCI-H1975 cells and PC-9 cells were used as vimentin-positive and vimentin-negative model cells, respectively. Human lung adenocarcinoma cells NCI-H1975 cells and PC-9 cells were seeded at a density of 200,000 cells / well. In a six-well plate, after the cells adhere to the wall, wash the cells with buffer solution, and incubate them with 200μl 10 μM vim-TPE-1 and vim-TPE-2 at room temperature for 2h respectively, and observe the green color of the cells under a 40× field of view with a fluorescence microscope Fluorescence brightness ( Figure 14 , 15 ). The fluorescence imaging results of the two kinds of tumor cells showed obvious differences: NCI-H1975 tumor cells showed obvious green fluorescence, while PC-9 tumor cells had almost no fluorescence, indicating that vim-TPE-1 and vim-TPE-2 can distinguish EMT This result proves the appli...

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Abstract

The invention discloses a waveform protein response type wash-free fluorescent probe as well as a preparation method and application thereof. The probe comprises a peptide fragment with affinity to waveform protein, an aggregation-induced luminescent type fluorescent group and a connecting group for connecting the peptide fragment with the fluorescent group. The probe can specifically generate thefluorescence response to the waveform protein and has advantages of high simplicity and convenience in operation, washing prevention, low detection cost, high specificity, high accuracy and the like.The probe serving as a detection reagent can be used for specific detection of epithelial-mesenchymal transition type tumor cells and tumor tissues with high sensitivity.

Description

Technical field [0001] The invention relates to the field of biological detection, in particular to a vimentin-responsive no-wash fluorescent probe, and a preparation method and application thereof. Background technique [0002] Vimentin is a classic biomarker of epithelial-mesenchymal transition. EMT is the key differentiation and transformation process of tissue morphogenesis in normal embryonic development. In tumorigenesis and development, EMT is considered to be an important potential reason for tumor stem cells to acquire invasiveness, metastasis ability and drug resistance. Therefore, in many types of tumors, the characteristics of EMT and the existence of tumor cells are closely related to tumor resistance, metastasis and recurrence. At present, studies have reported that small chemical molecules such as FiVe1 and Withaferin A, and nucleic acid aptamers such as NAS-24 and AptVim have an affinity or inhibitory effect on vimentin. [0003] In view of the important role of E...

Claims

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Application Information

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IPC IPC(8): G01N21/64C07K14/47C07K1/14C07K1/02
CPCC07K14/47G01N21/6428G01N21/6458
Inventor 魏涵于已芳田鑫张晓坚
Owner THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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