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Preparation for nanometer magnetic immunomicrosphere for enriching circulating cancer cells

A nano-magnetic and tumor cell technology, applied in the field of biomedicine, can solve the problems of unfavorable detection and low concentration of tumor cells, and achieve the effect of easy operation, strong magnetic response and small particle size

Inactive Publication Date: 2012-07-04
SHENZHEN ELDERLY MEDICAL RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem that the concentration of tumor cells in the sample is extremely low in the detection of circulating tumor cells, which is not conducive to detection, and to provide a nano-magnetic immune microsphere for enrichment of circulating tumor cells and its use method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The preparation of embodiment 1 nanometer magnetic microsphere

[0021] Dissolve 100mg sodium alginate solution in 4ml pure water, add 2ml 5% FeC magnetic fluid solution, mix well and dissolve at 42°C, ultrasonic for 15min, and use 10% Na 2 CO 3 Adjust the pH of the mixture to about 10, and raise the temperature to 60°C. Under the conditions of ultrasonic and high-speed stirring, the mixed solution was added dropwise into 90ml of AOT / n-heptane oil phase at a temperature of 60°C to form a transparent gray-black reverse microemulsion system. 30% 4.5ml CaCl 2 The solution is added into the microemulsion system, after magnetic separation, washed with acetone, ethanol and distilled water respectively, then centrifugally sorted, vacuum freeze-dried and stored.

Embodiment 2

[0022] Example 2 Preparation of Magnetic Microsphere-Antibody Conjugate

[0023] Take 1mg of the prepared magnetic microspheres, wash with phosphate buffer for 3 times, dilute to 4ml with 0.01mol / L PBS pH7.0, add 5mg of water-soluble EDC and 7.5mg of suflo-NHS, mix well, and keep at room temperature After reacting for 15 min, 50 mg of 6-aminocaproic acid was added, and after stirring at room temperature for 3 h, 500 μl (40 μg / ml) of EpCAM monoclonal antibody (BD Pharmingen, USA) was added, stirred gently for 6 h, and mixed with 0.2 M glycine solution (containing 0.2 %BSA) 1ml for sealing, magnetic separation and washing, adding storage solution and storing at 4°C.

[0024] Observed by transmission electron microscope, the microspheres are spherical and have good dispersion, the particle size distribution is between 45-89nm, and the average particle size is 56nm. The protein content in the nano-immunomagnetic microspheres was determined by Bradford assay, and 108.6 μg of antib...

Embodiment 3

[0025] Example 3 Enrichment and detection of breast cancer tumor cells

[0026] Add an appropriate amount of nano-immunomagnetic microspheres to each sample, mix well, rotate and stir at room temperature for 30 minutes, separate with a magnetic separator, wash the magnetic microspheres with PBS or saline several times to remove impurities, and finally resuspend the magnetic microspheres in an appropriate In the liquid, concentrated, pure circulating tumor cells are obtained.

[0027] The preparation of embodiment 3 streptavidin immunomagnetic microspheres

[0028] Take 1mg of the prepared magnetic microspheres, wash with phosphate buffer for 3 times, dilute to 4ml with 0.01mol / L PBS pH7.0, add 5mg of water-soluble EDC and 7.5mg of suflo-NHS, mix well, and keep at room temperature React for 15 minutes, then add 50 mg of 6-aminocaproic acid, rotate and stir at room temperature for 3 hours, add 500 μl (40 μg / ml) streptavidin, stir gently for 6 hours, and seal with 1ml of 0.2M gl...

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Abstract

The invention belongs to the technical field of biomedicine and discloses a nanometer magnetic immunomicrosphere for enriching circulating cancer cells and a use method thereof. A nanometer carbon coated pure ferromagnetic microsphere (Fe@C) is taken as an inner core and is connected with a cancer cell antibody, so as to prepare the nanometer magnetic immunomicrosphere for enriching the circulating cancer cells. By utilizing the magnetic immunomicrosphere to enrich the circulating cancer cells, the sensitivity of the detection for the circulating cancer cells is efficiently increased. The immunomicrosphere has the characteristics of strong magnetic reactivity, easiness in operation, and the like, and is applied to the quick enriching of the circulating cancer cells.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the preparation and application method of nanometer magnetic immune microspheres enriched in circulating tumor cells. technical background [0002] According to the pathway of tumor cell micrometastasis, tumor cells circulating in the blood or lymphatic vessels are called circulating tumor cells (circulating tumor cells, CTCs), and cell clumps formed by the aggregation of several circulating tumor cells are called circulating tumor microemboli ( Circulating tumor microemboli, CTM). CTM is the "collective migration" behavior of tumor cells, which has higher metastatic potential because of its ability to resist apoptosis and maintain cell proliferation. Due to the very small amount of circulating tumor cells in blood or lymph fluid and the lack of effective specific markers, the research on CTC has been slow. The existing separation and detection techniques have their own a...

Claims

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Application Information

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IPC IPC(8): B01J20/28G01N1/40G01N33/574
Inventor 李富荣齐晖周汉新
Owner SHENZHEN ELDERLY MEDICAL RES INST
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