A nano-microsphere time-resolved fluorescent probe and its preparation method and application

A time-resolved fluorescence, nano-microsphere technology, applied in the field of detection, can solve the problem of insufficient fluorescence intensity of rare earth element fluorescent probes, and achieve the effects of large compatibility of detection samples, rapid and quantitative combination, and high fluorescence intensity

Active Publication Date: 2016-12-14
上海溯源生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is that existing rare earth element fluorescent probes have defects such as insufficient fluorescence intensity.

Method used

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  • A nano-microsphere time-resolved fluorescent probe and its preparation method and application
  • A nano-microsphere time-resolved fluorescent probe and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 Preparation of carboxylated polystyrene nanospheres

[0022] Dissolve 10mm of styrene monomer and 0.95mm of acrylic acid monomer in 10ml of deionized water containing 0.45mm of sodium dodecylsulfonate, add it to a round bottom flask, stir evenly with a magnetic stirring bar, and then use high-purity nitrogen gas to Remove all the air in the bottom flask, seal and heat to 70°C, add 0.5ml of 0.15mm potassium persulfate, seal and stir for 8 hours in an oxygen barrier, then cool down to room temperature, then filter the reaction solution with whatman 2v filter paper (pore size 8 μm), Finally, a dialysis bag (molecular weight cut-off: 30,000 Da) was used for dialysis against deionized water for 5 days, and the liquid in the dialysis bag was collected and stored at 4° C. by adding 0.05% sodium azide.

[0023] By measurement, the prepared carboxylated polystyrene nanospheres have a diameter of 190 ± 10nm, a surface charge Surface Charge (μeq / g) of 170-200, and a c...

Embodiment 2

[0024] The preparation of embodiment 2 nano fluorescent microspheres

[0025] Get a small amount of 190nm polystyrene microspheres prepared in Example 1, add in 10ml of deionized water and acetone mixed solution (v / v=1:1), make the density of polystyrene microspheres in the reaction solution be about 1 ×10 14 Each, stir well, add 100ul 0.1M europium trichloride, 1 μl 0.1M terbium trichloride, 400 μl 0.1M β-diketone (β-NTA), 300 μl trioctylphosphine oxide (TOPO), 100 μl phenanthroline, First heat to 60°C and stir at constant temperature to avoid light for 10 hours, then lower to room temperature and react for 2 hours, finally remove the organic solvent in the solution by vacuum distillation, dialyze against deionized water for 5 days, remove the remaining small molecular substances, collect and dialyze The liquid in the bag was stored at 4°C by adding 0.05% sodium azide.

[0026] Through testing and calculation, the average number of europium ion chelates wrapped in each nano...

Embodiment 3

[0027] Embodiment 3 Comparison of fluorescence intensity of nano-fluorescent microspheres by different wrapping methods

[0028] Refer to the formula and steps of Example 2 to prepare undoped terbium ions, doped terbium ions without using phenanthroline, and undoped terbium ions using phenanthroline nano-fluorescent microspheres, and compared the fluorescence intensity. The results are shown in Table 1 .

[0029] Table 1

[0030]

[0031] Note: (1) Fluorescence intensity is defined as the multiple of the fluorescence intensity produced by a nanosphere after excitation that is equivalent to that of a single free europium ion chelate;

[0032] (2) Commercial fluorescent microspheres with a particle size of 0.2 μm were purchased from Thermo Fisher Scientific under the trade name of Fluoro-Max Carboxylate-Modified and Streptavidin-Coated Europium Chelate Particles.

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Abstract

The invention belongs to the technical field of detection, and in particular relates to a nano-microsphere time-resolved fluorescent probe, a preparation method and application thereof. The invention discloses a nano-microsphere time-resolved fluorescent probe, which is a nano-microsphere coated with a rare earth element fluorescent complex by a macromolecular material, and is characterized in that the rare earth element fluorescent complex includes substances in the following molar ratio: rare earth element ions: β-diketone chelating agent: fluorescence enhancement synergist = 1:4:5, wherein the rare earth element ion is a double-doped mixture of Eu3+ and other lanthanide ions in a molar ratio of 100:1-1000:1.

Description

technical field [0001] The invention belongs to the technical field of detection, and in particular relates to a nano-microsphere time-resolved fluorescent probe, a preparation method and application thereof. Background technique [0002] Time resolved fluoroisnmunoassay (TRFIA) is a relatively new detection method proposed in the 1980s. TRFIA uses rare earth ions as tracers to label proteins, polypeptides, hormones, antibodies, nucleic acid probes or biological Active cells, their chelating agents, enhancing solutions (some are not required) in the system to be reacted (such as: antigen antibody immune reaction, biotin avidin reaction, nucleic acid probe hybridization reaction, killing reaction of target cells and effector cells, etc.) After the reaction occurs, the fluorescence intensity in the final product is measured with a time-resolved fluorometer, and the concentration of the analyte in the reaction system is estimated according to the ratio of the fluorescence inten...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09K11/06C09K11/02G01N21/64G01N33/52
Inventor 肖理文张玲郭文豪王立梁俊张琪
Owner 上海溯源生物技术有限公司
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