Method for tumor cell detection through porous gold nanospheres

Abstract

The invention belongs to the field of tumor cytology, and discloses a method for tumor cell detection through porous gold nanospheres. The method comprises the following steps: (1) preparing the porous gold nanospheres; (2) carrying out co-incubation on the porous gold nanospheres and a sulfydryl-modified aptamer to prepare functionalized porous gold nanospheres; (3) binding the functionalized porous gold nanospheres with sample cells, and removing the unbound porous gold nanospheres, so that a cell sample to be detected is obtained; and (4) adding a bicarbonate solution into the cell sample to be detected, and carrying out illumination under a 808nm laser. The method for the tumor cell detection through the porous gold nanospheres has the advantages that the operation is simple, and the detection cost is low; and the adopted porous gold nanospheres can be simply prepared, achieve high light and heat stability and high biocompatibility, and can produce a high temperature under the illumination of the 808nm laser for thermal decomposition of bicarbonate, so that air pressure changes in a reaction system can be monitored to distinguish tumor cells from normal cells.

Description

technical field

[0001] The invention belongs to the field of tumor cytology, and in particular relates to a method for detecting tumor cells by using porous gold nanospheres. Background technique

[0002] Tumor is a new organism formed by the body under the action of various carcinogenic factors when a certain cell in a local tissue loses its normal regulation of its growth at the gene level, resulting in abnormal clonal proliferation. At present, the treatment of cancer is mainly divided into traditional Chinese medicine treatment, surgical treatment, radiotherapy and chemotherapy. Regardless of the type of treatment, the earlier the cancer is detected, the greater the chances of successful cancer treatment. Therefore, early warning and early diagnosis of tumors have important clinical significance. At present, the detection of tumor cells mainly uses fluorescence analysis, enzyme-linked immunosorbent assay, flow cytometry and other biosensing methods. However, the above...

Examples

Embodiment 1

[0033] Preparation of porous gold nanospheres:

[0034] 1) Preparation of gold-silver composite nanospheres: Weigh 0.18g of polyvinylpyrrolidone powder and disperse it into a round-bottomed flask containing 18mL of secondary water (note: add small amounts several times to ensure that the polyvinylpyrrolidone added each time is completely dissolved) Add the next time), under the dark condition, add hydroquinone solution (360μL, 50mM), silver nitrate solution (40μL, 60mM), chloroauric acid solution (640μL, 25mM) in order, at 37 ℃, shaking table After shaking vigorously for 5 minutes, the solution was left to stand for 90 minutes, and the color of the solution gradually changed from light yellow to blue, and finally to light red, indicating that gold-silver composite nanospheres had been successfully prepared;

[0035] 2) Dissolution of silver atoms: After adding 200 μL of concentrated ammonia water to the gold-silver composite nanosphere solution, shake it on a shaker at 37°C fo...

Embodiment 2

[0038] Adopt embodiment 1 to prepare porous gold nanosphere;

[0039] Determination of air pressure value: In a 0.6mL centrifuge tube, mix 50μL of ammonium bicarbonate solution (1M) and 0.75mg / mL porous gold nanospheres in equal volumes, then insert a portable air gauge and seal the centrifuge tube, and the air pressure will be measured under 808nm laser After irradiating for 10 minutes, record the air pressure in the centrifuge tube.

[0040] figure 2 It is the histogram of pressure value change (△P) under different experimental conditions, represented by figure 2 It can be seen that the porous gold nanospheres can decompose the ammonium bicarbonate solution to generate gas under 808nm laser irradiation, resulting in an increase in the pressure value; when there is no porous gold nanospheres or 808nm laser irradiation, the reaction system will not produce gas; no carbonic acid is added When the ammonium hydrogen solution is added, the porous gold nanospheres convert the a...

Embodiment 3

[0042] The invention provides a method for detecting tumor cells by using porous gold nanospheres, comprising the following steps:

[0043] (1) adopt embodiment 1 to prepare porous gold nanosphere;

[0044] (2) Co-incubate the porous gold nanospheres with the thiol-modified aptamer: add 32 μL of 100 μM thiol-modified MUC1 aptamer (sequence 1: 5'-HS-tttttttttt gcagttgatc ctttggatac cctgg-3', thiol is HS indicated) and 500 μL porous gold nanospheres (0.75 mg / mL) were slightly shaken overnight, then gradually added 5M sodium chloride (2 μL every two hours, a total of 5 times, namely 10 μL), stood overnight, and centrifuged at 5000 r / pm After 5 minutes, remove 90% of the supernatant, then add 450 μL of secondary water, repeat the centrifugation operation 3 times, and set the volume to 500 μL to obtain functionalized porous gold nanospheres;

[0045] (3) Combine functionalized porous gold nanospheres with sample cells: mix the cell culture medium containing 10 μL of MUC1 aptamer-m...

technical field

[0001] The invention belongs to the field of tumor cytology, and in particular relates to a method for detecting tumor cells by using porous gold nanospheres. Background technique

[0002] Tumor is a new organism formed by the body under the action of various carcinogenic factors when a certain cell in a local tissue loses its normal regulation of its growth at the gene level, resulting in abnormal clonal proliferation. At present, the treatment of cancer is mainly divided into traditional Chinese medicine treatment, surgical treatment, radiotherapy and chemotherapy. Regardless of the type of treatment, the earlier the cancer is detected, the greater the chances of successful cancer treatment. Therefore, early warning and early diagnosis of tumors have important clinical significance. At present, the detection of tumor cells mainly uses fluorescence analysis, enzyme-linked immunosorbent assay, flow cytometry and other biosensing methods. However, the above...

Examples

Embodiment 1

[0033] Preparation of porous gold nanospheres:

[0034] 1) Preparation of gold-silver composite nanospheres: Weigh 0.18g of polyvinylpyrrolidone powder and disperse it into a round-bottomed flask containing 18mL of secondary water (note: add small amounts several times to ensure that the polyvinylpyrrolidone added each time is completely dissolved) Add the next time), under the dark condition, add hydroquinone solution (360μL, 50mM), silver nitrate solution (40μL, 60mM), chloroauric acid solution (640μL, 25mM) in order, at 37 ℃, shaking table After shaking vigorously for 5 minutes, the solution was left to stand for 90 minutes, and the color of the solution gradually changed from light yellow to blue, and finally to light red, indicating that gold-silver composite nanospheres had been successfully prepared;

[0035] 2) Dissolution of silver atoms: After adding 200 μL of concentrated ammonia water to the gold-silver composite nanosphere solution, shake it on a shaker at 37°C fo...

Embodiment 2

[0038] Adopt embodiment 1 to prepare porous gold nanosphere;

[0039] Determination of air pressure value: In a 0.6mL centrifuge tube, mix 50μL of ammonium bicarbonate solution (1M) and 0.75mg / mL porous gold nanospheres in equal volumes, then insert a portable air gauge and seal the centrifuge tube, and the air pressure will be measured under 808nm laser After irradiating for 10 minutes, record the air pressure in the centrifuge tube.

[0040] figure 2 It is the histogram of pressure value change (△P) under different experimental conditions, represented by figure 2 It can be seen that the porous gold nanospheres can decompose the ammonium bicarbonate solution to generate gas under 808nm laser irradiation, resulting in an increase in the pressure value; when there is no porous gold nanospheres or 808nm laser irradiation, the reaction system will not produce gas; no carbonic acid is added When the ammonium hydrogen solution is added, the porous gold nanospheres convert the a...

Embodiment 3

[0042] The invention provides a method for detecting tumor cells by using porous gold nanospheres, comprising the following steps:

[0043] (1) adopt embodiment 1 to prepare porous gold nanosphere;

[0044] (2) Co-incubate the porous gold nanospheres with the thiol-modified aptamer: add 32 μL of 100 μM thiol-modified MUC1 aptamer (sequence 1: 5'-HS-tttttttttt gcagttgatc ctttggatac cctgg-3', thiol is HS indicated) and 500 μL porous gold nanospheres (0.75 mg / mL) were slightly shaken overnight, then gradually added 5M sodium chloride (2 μL every two hours, a total of 5 times, namely 10 μL), stood overnight, and centrifuged at 5000 r / pm After 5 minutes, remove 90% of the supernatant, then add 450 μL of secondary water, repeat the centrifugation operation 3 times, and set the volume to 500 μL to obtain functionalized porous gold nanospheres;

[0045] (3) Combine functionalized porous gold nanospheres with sample cells: mix the cell culture medium containing 10 μL of MUC1 aptamer-m...

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