Bioassay probe for early diagnosis of bacterial infection

A technology of fluorescent probes and fluorescent dyes, applied in the synthesis and application of traceable galactosylated near-infrared fluorescent probes, can solve problems such as poor specificity, inability to target diagnosis, inability to perform in vivo diagnosis, etc., and achieve good applications Foreground effect

Active Publication Date: 2014-07-02
CHINA PHARM UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0003] FDG / PET method (The Journal of Nuclear Medicine.2001,42(9):1412-1417; Annals of Nuclear Medicine.1996,10(2):185-191) is one of the methods for early diagnosis of bacterial infectious diseases. However, its biggest disadvantage is poor specificity and cannot be targeted for diagnosis
The In-WBC method for labeling leukocytes (Bioorganic & Medicinal Chemistry Letters.2012,22(8):2833-2836) has the disadvantage that in vivo diagnosis cannot be performed, and leukocytes must be extracted from the body
[0007] At present, there is no report on the specific combination of galactosylated near-infrared fluorescent probes and maltose-binding proteins (MBPs) in the targeted diagnosis of various bacterial infections at home and abroad.

Method used

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  • Bioassay probe for early diagnosis of bacterial infection
  • Bioassay probe for early diagnosis of bacterial infection
  • Bioassay probe for early diagnosis of bacterial infection

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Synthesis of Gal-MPA

[0034] MPA activation: Dissolve 5.0mg of MPA in 2.0mL of dimethylformamide (DMF), add 4.8mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) 0.6 mg, stirred at room temperature overnight in the dark.

[0035] Synthesis of Gal-MPA: 1.1 mg of galactosamine was dissolved in 2.0 mL of pyridine, added dropwise to the above MPA activation solution, and stirred overnight at room temperature in the dark. Freeze and rotary evaporate to remove pyridine. Preliminary identification of reaction products by thin layer chromatography. The reaction product was dialyzed, purified on Sephadex G10, and freeze-dried to obtain Gal-MPA. A certain amount of Gal-MPA dissolved in distilled water was characterized by ultraviolet absorption and fluorescence emission spectra, which showed that it had stable optical properties. UV absorption spectrum see figure 1 , the fluorescence emission spectrum see figure 2 . ...

Embodiment 2

[0037] cell culture

[0038] Human cell line L02 (normal human hepatocytes) was incubated at 37°C in an incubator containing 5% carbon dioxide. Cultured in RPMI1640 supplemented with 10% FBS, 100 μg / ml penicillin and 100 μg / ml streptomycin.

Embodiment 3

[0040] Cytotoxicity

[0041] 5) Cell proliferation experiments were performed with L02 cells. Cells were seeded into 96-well plates (1×10 4 cells / well) for 24 hours under culture conditions.

[0042] 6) Gal-MPA solution dissolved in phosphate buffered PBS was added to the cells, and the culture was continued for 24 hours, and the sample concentration ranged from 1.5625 to 100 μg / mL.

[0043] 7) Add 10 μl thiazolium blue (MTT) solution (5.0 mg / mL) to each well and incubate for another 4 hours. Carefully remove residual MTT in the medium, and dissolve the purple crystals in 150 μL dimethyl sulfoxide (DMSO).

[0044] 8) After shaking and mixing for ten minutes, all test samples were detected by ELIASA, and the cell viability was calculated by the following formula: Cell survival rate = (average absorbance of sample group - average absorbance of medium wells) / (average absorbance of solvent group - medium The average absorbance of the well) × 100%. See image 3 , Gal-MPA wa...

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Abstract

The invention relates to the field of synthesis and application of a bioassay probe for early diagnosis of bacterial infection and in particular relates to synthesis and application of a traced galactosylated near-infrared fluorescent probe which can specifically target various bacteria. A galactosylated compound is prepared by a covalent linkage method by using D-galactosamine hydrochloride and a near-infrared fluorescent dye-indocyanine green as main raw materials, so that the galactosylated compound has a targeted diagnosis effect on bacteria. Experiments prove that the synthesized probe is safe and effective, can quickly reach the bacterially infected parts of living bodies, can serve as an assay probe of various bacterial infections, and has good application prospects. If connected with antibiotics, the probe can target the drugs towards the bacterially infected parts to directly kill the bacteria and reduce toxic and side effects on normal tissues and cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the synthesis and application of live detection probes for the early diagnosis of bacterial infection, and to the synthesis and application of traceable galactosylated near-infrared fluorescent probes that can specifically target a variety of bacteria. application. Background technique [0002] According to the statistics of the World Health Organization, 25% of the deaths in the world are caused by bacterial infectious diseases (Journal of Clinical Investigation, 2003, 111(9): 1265-1273). Although antibiotic treatment has a good effect on killing pathogenic bacteria, it will also reduce the body's immunity and induce superinfection and side effects (Chinese Journal of Biochemical Medicine, 2012, 33(3):326-328). What's more serious is that at present, whether it is bacterial inflammation or not, antibiotics are used, which not only delays the treatment of the disease, but also induc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06A61K49/00
Inventor 顾月清马宇翔刘翠翠尹德艳陈海燕王娜苏杉雨涵李瑛张敏冯松
Owner CHINA PHARM UNIV
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