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Application of a near-infrared xanthene fluorescent dye in fluorescent labeling of Schistosoma japonicum adults

A fluorescent dye and fluorescent labeling technology is applied in the application field of near-infrared xanthene fluorescent dyes in the fluorescent labeling of schistosomiasis adults, and can solve the problem of inability to long-term fluorescent observation, fluorescent interference, poor hydrophilicity of organic small molecule fluorescent dyes, etc. problem, to achieve the effect of good light penetration performance and high signal-to-noise ratio

Active Publication Date: 2020-08-21
GANNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, traditional organic fluorescent dyes, such as resorufin, were used as fluorescent probes to study adults of Schistosoma japonicum in the above studies, which had the following disadvantages: (1) short-wavelength excitation and emission had biological spontaneous background fluorescence interference; (2) large Some fluorescent probes have poor photostability, which prevents long-term fluorescence observation; (3) Most organic small molecule fluorescent dyes have poor hydrophilicity, which limits their biological applications
It can be seen that there are contradictions between the fluorescent dyes in the prior art and the physiological structure characteristics of adults of Schistosoma japonicum, which lead to problems such as poor fluorescent dyeing effect, large background fluorescence interference and poor fluorescence penetration in the fluorescence imaging process of Schistosoma japonicum adults.

Method used

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  • Application of a near-infrared xanthene fluorescent dye in fluorescent labeling of Schistosoma japonicum adults
  • Application of a near-infrared xanthene fluorescent dye in fluorescent labeling of Schistosoma japonicum adults
  • Application of a near-infrared xanthene fluorescent dye in fluorescent labeling of Schistosoma japonicum adults

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Mix 5.33g (40mmol) of 2-methylbenzoxazole with 20mL of toluene, then add 6.86g (44mmol) of ethyl iodide, and react at 120°C for 24h; cool to room temperature, and filter under reduced pressure to obtain a crude extract. Washed three times with toluene (10 mL×3)) to obtain compound 2 with a yield of 85%. 1 H NMR (400MHz, MeOD) δ8.19–7.93 (m, 2H), 7.80 (dd, J = 9.3, 5.5Hz, 2H), 4.67 (q, J = 7.4Hz, 2H), 3.13 (s, 3H) ,1.61(s,3H); Exact Mass: C 10 h 12 NO + , 162.0913, ESI-MS: m / z, 162.1.

[0059] Compound 22.89g (1mmol) and N,N-diphenylformamidine 1.93g (1mmol) were mixed, and a solid-phase melting reaction was carried out at 140°C for 0.5h; a mixed solvent of dichloromethane and methanol was used as the eluent ( The volume ratio of dichloromethane and methanol in the eluent is 20:1) The obtained crude product was separated by column chromatography to obtain compound 3 with a yield of 65%. 1 H NMR (400MHz, CDCl 3 )δ11.72(d,J=14.0Hz,1H),8.64(dd,J=14.1,12.1Hz,1H),7.61(d...

Embodiment 2

[0063] The near-infrared xanthene fluorescent dye (NIR-RB) having the structure shown in formula I is carried out ultraviolet absorption spectrum and fluorescence spectrum test, specifically as follows:

[0064] Mix NIR-RB with ethanol to obtain 1mmol / L NIR-RB ethanol mother liquor, then dilute with ethanol to obtain 10μmol / L NIR-RB dilution, carry out ultraviolet absorption spectrum and fluorescence spectrum on the NIR-RB dilution In the test, the scanning range of the ultraviolet absorption spectrum measurement is 500-850nm; the excitation wavelength of the fluorescence spectrum measurement is 620nm, and the emission spectrum range is 640-850nm.

[0065] figure 2 It is the ultraviolet absorption spectrum and the fluorescence spectrum of the NIR-RB dilution, by figure 2 It can be seen that the emission peak of NIR-RB is located in the near-infrared region of 650-850 nm.

Embodiment 3

[0067] The cell fluorescence imaging of NIR-RB was detected, as follows:

[0068] (1) Reagents and materials

[0069]Dimethyl sulfoxide (AR) was purchased from Aladdin Chemical Reagent Co., Ltd., RPMI 1640 culture medium was purchased from Thermo Fisher Scientific, and human cervical cancer cells (HeLa) were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Place.

[0070] (2) Fluorescence imaging of human cervical cancer cells

[0071] Human cervical cancer cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C, 5% CO 2 and 95% air atmosphere; under the condition of excitation wavelength of 635nm, the fluorescent labeling of cultured human cervical cancer cells was detected by OLYMPUS FV1000 laser confocal fluorescence microscope, and the fluorescent signal of 650-750nm was collected Perform fluorescence imaging.

[0072] image 3 Fluorescent imaging of human cervical cancer cells, by image 3 It ...

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Abstract

The invention provides application of near infrared xanthene fluorescent dye in fluorescent labeling of adult schistosoma. The near infrared xanthene fluorescent dye has the structure shown in formulaI. In the application, the near infrared xanthene fluorescent dye (NIR-RB) with the structure of the formula I has an emission peak located in a near infrared region and has good light transmittancein living body fluorescence imaging. The NIR-RB can perform fluorescent labeling imaging on the adult schistosomiasis; meanwhile, the fluorescence intensity of the adult schistosoma is quantitativelyanalyzed, the signal-to-noise ratio (S / N) of a fluorescent signal in a worm body region and a background region is 10, which indicates that the NIR-RB used for the fluorescence imaging of the adult schistosoma has a high signal-to-noise ratio.

Description

technical field [0001] The invention relates to the technical field of bio-optical labeling, in particular to the application of a near-infrared xanthene fluorescent dye in the fluorescent labeling of schistosomiasis adults. Background technique [0002] Schistosomiasis is an infectious disease that is prevalent in tropical and subtropical regions, seriously endangering people's health and affecting social and economic development. Schistosomiasis is endemic in 74 countries and regions. There are currently about 652 million people in the world at risk, 193 million infected people, and about 120 million symptomatic cases, of which 20 million are severe cases. China is the main endemic area of ​​schistosomiasis. [0003] Fluorescence imaging can be used in the study of schistosomiasis, Andrea et al. ,113,130-133) used fluorescent probes to detect the content of nitric oxide in Schistosoma japonicum, and found that the release of nitric oxide in worms was dependent on nitric o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 吴勇权曾红范小林李勋郭维
Owner GANNAN NORMAL UNIV
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