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Near-infrared two-window fluorescence immunoprobe as well as preparation method and application thereof

A fluorescence immune and near-infrared technology, applied in the field of fluorescence imaging, can solve the problems of complex composition and poor stability, and achieve the effects of low light scattering, low cost and low dosage.

Active Publication Date: 2021-11-23
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A small number of immunoprobes conjugated with antibodies are mostly physically mixed, resulting in complex components and poor stability, which are not suitable for clinical use

Method used

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  • Near-infrared two-window fluorescence immunoprobe as well as preparation method and application thereof
  • Near-infrared two-window fluorescence immunoprobe as well as preparation method and application thereof
  • Near-infrared two-window fluorescence immunoprobe as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] The preparation method of a typical near-infrared fluorescent dye is as follows: image 3 shown, including the following steps:

[0087] (1) Preparation of intermediate (2)

[0088] Dissolve compound (1) in solvent 1 (toluene, benzene, dimethyl sulfoxide, N,N-dimethylformamide or N,N-dimethylacetamide, etc.), add dropwise In the eggplant-shaped bottle of lactone, the reaction temperature is 40-120° C. (preferably 110° C.), and the reaction time is preferably 2 hours to obtain the intermediate (2).

[0089] (2) Preparation of intermediate (3)

[0090] Dissolve the intermediate (a) and base in the solvent, add them in batches to the eggplant-shaped bottle containing the intermediate (2) and stir, the reaction temperature is 40-90°C (preferably 80°C), and the reaction time is preferably 60 minutes , to obtain intermediate (3). The base is selected from various organic bases or inorganic bases, preferably sodium carbonate, sodium bicarbonate, potassium carbonate, potass...

Embodiment 2

[0102] Preparation of conjugates of near-infrared fluorescent dyes and single-domain antibodies figure 2 The steps shown are carried out as follows:

[0103] (1) After the single domain antibody and the reducing agent are mixed, shake and react for a period of time, the near-infrared fluorescent dye is dissolved in an organic solvent, and added to the mixed solution containing the antibody and the reducing agent in batches to obtain a mixture. The reducing agent used to break the disulfide bonds of the antibody is selected from various organic and inorganic reducing agents, preferably tris(2-chloroethyl)phosphate, diethyltriaminepentaacetic acid, 5,5'-dithiobis (2-nitrobenzoic acid). The organic solvent is selected from N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, etc., the reaction temperature is 4-60°C, preferably 37°C, and the reaction time is preferably 60 minutes.

[0104] (2) Add the mixture obtained in step (1) to the desalting column. Collect f...

Embodiment 3

[0106] A kind of preparation of near-infrared fluorescent dye ICGM according to Figure 4 The steps shown are carried out as follows:

[0107] (1) Preparation of intermediate (2)

[0108] Compound (1) (2.09g, 10mmol) was dissolved in 5mL of toluene, added dropwise into an eggplant-shaped bottle filled with 1,3-propane sultone, reacted at 110°C for 8 hours, collected the dark gray solid by filtration, and used 20 mL of chloroform was washed three times to obtain intermediate (2).

[0109] (2) Preparation of intermediate (3)

[0110] Intermediate (2) (3.31 g, 10 mmol) and biguanidine glutamate hydrochloride (2.85 g, 10 mmol) were added to a 50 mL round bottom flask containing acetic anhydride (3 mL) and potassium acetate (0.98 g, 10 mmol). The whole mixture was heated to 70°C for 1 hour, cooled to room temperature, and poured into saturated sodium bicarbonate solution. The red precipitate was washed with ether. The final product is eluent CH 2 Cl 2 / MeOH (20 / 1; v / v) was s...

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Abstract

The invention relates to a near-infrared two-window fluorescence immunoprobe as well as a preparation method and application thereof. The fluorescence immunoprobe is a conjugate of near-infrared fluorescent dye and a single-domain antibody. The preparation method of the fluorescence immunoprobe comprises the steps of 1, mixing a single-domain antibody and a reducing agent, then conducting oscillatory reaction, and then adding the near-infrared fluorescent dye dissolved in an organic solvent; and 2) adding the mixture in the step (1) into a desalting column, removing the residual near-infrared fluorescent dye, and then centrifugally collecting supernate. Compared with the prior art, the fluorescence immunoprobe has the advantages that the maximum absorption and emission of the fluorescence immunoprobe are in a first near-infrared window (650-900nm) region, and the fluorescence intensity in a second near-infrared window (1000-1700nm) region is greatly enhanced after the fluorescence immunoprobe is combined with an antigen, so that the fluorescence immunoprobe has relatively low light scattering, relatively high in-vivo imaging resolution, relatively deep penetration depth and relatively low dosage; and the fluorescence immunoprobe can be applied to marking, tracking and living body imaging of biomacromolecules.

Description

technical field [0001] The invention belongs to the technical field of fluorescence imaging, and relates to a near-infrared two-window fluorescence immunoprobe, a preparation method and application thereof. Background technique [0002] Immunoprobe refers to a conjugate that labels an antibody with a marker with special physical and chemical properties, so that the antigen it recognizes can be qualitatively and quantitatively detected. Currently, labels used to prepare immunoprobes include radioisotopes, colloidal gold, fluorescent proteins, and organic fluorescent dye molecules. Among them, fluorescent immunoprobes prepared using organic fluorescent dye molecules are widely used in the labeling, imaging and tracking of biological macromolecules due to their high sensitivity, high safety, and real-time monitoring. [0003] However, most of the biomacromolecules used for specific targeting are monoclonal antibodies with relatively large molecular weight and weak tissue or tu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D403/14C09K11/06G01N21/64
CPCC07D403/14C09K11/06G01N21/6428C09K2211/1029
Inventor 史逸冰应天雷吴艳玲
Owner FUDAN UNIV
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