Fluorescence probe and synthetic method thereof and application of fluorescence probe in circulating tumor cell detection

A technology for fluorescent probes and tumor cells, applied in fluorescence/phosphorescence, measuring devices, and material analysis through optical means, can solve problems such as complex operations, high costs, and poor accuracy, and achieve improved sensitivity, short time consumption, and The effect of low instrument requirements

Inactive Publication Date: 2017-11-17
THE FIFTH AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to: overcome the problems of high cost, complicated operation and poor accuracy in the existing tumor cell detection, and provide a fluorescent probe with high sensitivity, simple and fast operation, low cost and its synthesis method and application

Method used

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  • Fluorescence probe and synthetic method thereof and application of fluorescence probe in circulating tumor cell detection
  • Fluorescence probe and synthetic method thereof and application of fluorescence probe in circulating tumor cell detection
  • Fluorescence probe and synthetic method thereof and application of fluorescence probe in circulating tumor cell detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Synthesis of a 2-fold amplified fluorescent probe using the Tokyo Green fluorophore as an example

[0039]The double-amplified fluorescent probe mainly includes four parts: a phenylalanine-lysine protecting group, a self-degradation bond, a fluorescent group Tokyo Green, and a folic acid molecule that targets CTC.

[0040] Firstly, phenylalanine ester (1a) protected by benzyloxycarbonyl reacts with lysine (1b) protected by di-tert-butyl dicarbonate (PG1) to obtain product 1c lysyl phenylalanine ester. 1d diformyl chloride

[0041] 1c reacts with diacid chloride to form an acid anhydride intermediate, and then reacts with 1f fluorescent group-lysyl phenylalanine ester to obtain intermediate 1g with two fluorescent groups connected to Tokyo Green.

[0042] Use trifluoroacetic acid to remove the Boc protecting group in compound 1g, and then react with folic acid in an amidating reagent such as EDCI / HOBt system to form 1g of a product coupled with folic acid, whi...

Embodiment 2

[0043] Example 2 CTC detection

[0044] Take 8 tubes of 5mL normal human anticoagulant blood, add different numbers of colon cancer cells and mix well, discard the supernatant after centrifuging at 1500rpm for 5min, add 1mL buffer solution containing 0.1% fluorescent probe prepared in Example 1, and warm at 37°C. After incubation for 10-20min, detect with a fluorescence spectrophotometer, the results are as follows: image 3 shown.

Embodiment 3

[0045] Example 3 Mouse peritoneal tumor development

[0046] Take 0.5 mL of the buffer solution containing 0.1% fluorescent probe prepared in Example 1, inject it into the abdominal cavity of the mouse, anesthetize the mouse with barbiturate 20 minutes later, fix the limbs, cut the abdominal cavity with ophthalmic scissors, and use 488 excitation light Observe the tumor under the LED light ( Figure 4 ).

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Abstract

The invention discloses a fluorescence probe and a synthetic method thereof and an application of the fluorescence probe in circulating tumor cell detection. The fluorescence probe comprises a fluorescence group, a cathepsin B substrate and folic acid molecules, the fluorescence group is connected with the cathepsin B substrate through a self-degradation bond, and then is connected with the folic acid molecules; the fluorescence group is excited by the excitation light under a dissociation state, and then is enable covalent connection with other molecules for quenching. The fluorescence probe is the switching-mode fluorescence probe having a spontaneous cascade amplification function, can be used for avoiding complex operation steps of antibody or nucleic acid detection during a circulating tumor cell detection process, is used for an automatic biochemical analyzer as a matching reagent, is in favor of promoting automation detection of tumor cells, and has the advantages of simple and convenient operation, short time consuming, and low apparatus requirement. The fluorescence probe can be used for positioning minimal tumor cells and tubercle in clinical operation, and has wide application prospect and large market value.

Description

technical field [0001] The invention belongs to the technical field of biochemical detection, and more specifically relates to a fluorescent probe targeting cathepsin B, its synthesis method and its application in tumor detection. Background technique [0002] Tumor is the number one fatal disease in the world, and its morbidity and mortality have always been at the forefront of various diseases, and there has been a rapid growth trend in recent years. Due to the relatively insidious onset of tumors, metastasis has generally occurred when patients have clinical symptoms. According to statistics, more than 90% of cancer patients die of tumor metastasis. [0003] The number of tumor cells in peripheral blood is extremely small, and there is interference from a large number of normal cells, making detection difficult. At present, the commonly used detection techniques mainly include conventional cytological staining detection and immunocytochemical techniques. Most of these m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N21/64
CPCG01N33/574G01N21/6428G01N2021/6432
Inventor 方志远蒋白山
Owner THE FIFTH AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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