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Fluorescence imaging method for tumor cells

A fluorescence imaging and cell technology, used in fluorescence/phosphorescence, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problems of high price, complex cell imaging technology, insufficient sensitivity, etc. The effect of selectivity, broadening application fields and improving detection sensitivity

Pending Publication Date: 2020-12-22
QINGDAO UNIV OF SCI & TECH
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Problems solved by technology

The advantages brought by the present invention are that the detection sensitivity is improved to the greatest extent, the detection process is simplified, the detection cost is reduced, the application field is broadened, and the problems such as complicated living body and cell imaging technology, high price and insufficient sensitivity are solved. It has potential application value in the fields of in vivo and in vitro detection, in vivo and cell imaging; the second purpose of the present invention is to provide a method based on HAase-HA biorecognition, using hollow gold nanomaterials to load signal molecules for target cycle-autocatalytic cycle amplification The preparation method of the nanocomposite material of reaction; The third object of the present invention: provide a kind of nanocomposite material based on HAase-HA biorecognition function that utilizes hollow gold nanomaterials to carry out self-catalyzed cycle amplification reaction for HAase in tumor cells fluorescence imaging method

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  • Fluorescence imaging method for tumor cells

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Embodiment 1

[0023] A method for preparing a nanocomposite material for intracellular HAase fluorescence imaging proposed by the present invention, based on HAase-HA biorecognition, using hollow gold nanomaterials to carry signal molecules for self-catalytic cycle amplification reaction, comprising the following steps :

[0024] (1) Add 2 μL to 200 μL hollow gold nanocarrier solution to a final concentration of 1.0×10 -5 mol / L RhB solution, reacted on a shaker at 37°C for 12.0 hours, centrifuged, washed and stored for later use;

[0025] (2) Add 200 μL of 13 mg / mL carbonyldiimidazole in DMF solution to 0.5 mL of 100 mg / mL HA aqueous solution, react at room temperature for 2.0 h, add 100 μL of 25 mg NH 2 -Aqueous solution of PEG-SH and 10 μL trimethylamine solution, stirred overnight;

[0026] (3) Purified by dialysis membrane and freeze-dried to obtain complex HA-PEG-SH;

[0027] (4) Add 0.5mL 2.4mg / mL HA-PEG-SH aqueous solution to the nanocarrier composite prepared in step (1), and sti...

Embodiment 2

[0031] A method for fluorescence imaging of HAase in tumor cells by using hollow gold nanocarriers proposed by the present invention to construct nanocomposites based on HAase-HA biorecognition for self-catalyzed cycle amplification reaction, comprising the following steps:

[0032] (1) Add the PBS dilution of the nanocomposite prepared in Example 1 to the Hela cell culture dish that has been cultured, and incubate at 37° C. for 2.0 h;

[0033] (2) As a control experiment, another Hela cell was incubated with 0.02g / mL HAase inhibitor 4-methylumbelliferone in advance for 2.0h before performing the same experiment;

[0034] (3) The experimental group and the control group were subjected to laser confocal scanning imaging, the excitation wavelength was selected to be 559nm, and the emission wavelength was 580-630nm.

[0035] Depend on figure 1 A, B can be seen, and the fluorescent signal in the Hela cells incubated with the nanocomposite material is clearly visible, indicating t...

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Abstract

The invention discloses a fluorescence imaging method for tumor cells. Hyaluronidase HAase is closely related to many malignant tumors. Researches show that the expression level of the HAase in cancercells is greatly increased. The invention provides a tumor cell fluorescence imaging method of HAase. According to the method, a hollow gold nano material is adopted to construct a nano composite material based on an HAase-HA biological recognition effect, and the nano composite material is subjected to an autocatalytic cyclic amplification reaction to be used for fluorescence imaging of the HAase in tumor cells. According to the method, a cell imaging system is optimized and improved, the sensitivity is improved to the greatest extent, the imaging process is simplified, the cost is reduced,the application field is widened, the problems of being complex in living body and cell imaging technologies, high in price, insufficient in sensitivity and the like are solved, more importantly, thecyclic amplification effect of signals can be obtained without any tool enzymes, and a new technology and method are provided for the fields of in-vitro and in-vivo detection, living bodies, cell imaging and the like.

Description

technical field [0001] The invention relates to fluorescence imaging of tumor cells, in particular to a fluorescence imaging method of hyaluronidase HAase in tumor cells, and belongs to the fields of photoelectrochemical biosensing analysis and biomedical clinical diagnosis. Background technique [0002] Bladder cancer is the most common malignant tumor of the male urinary system, and its morbidity and mortality both rank first in genitourinary system tumors. Because the traditional clinical diagnosis method of bladder cancer is mostly based on cystoscopy, not only trauma and pain, but also obviously insufficient for early clinical diagnosis. According to reports, hyaluronidase HAase is closely related to many malignant tumors. The enzyme was first isolated and purified by Frost in 1997. Studies have shown that the expression level of HAase is greatly increased in cancer cells, while the distribution in normal cells is less. In particular, the concentration of HAase was si...

Claims

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Application Information

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IPC IPC(8): C12Q1/34G01N21/64
CPCC12Q1/34G01N21/6486G01N21/6428G01N2021/6439G01N2333/928
Inventor 王卫李欣宋志灵
Owner QINGDAO UNIV OF SCI & TECH
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