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Nanometer europium fluorescent particle with performance of visible light excitation, preparation method and application thereof

A technology of fluorescent particles and visible light, which is applied in the field of preparation of nano-europium fluorescent particles, can solve the problems of easy leakage of fluorescent molecules, complicated labeling process, background fluorescence interference, etc., to prolong continuous measurement time, improve photobleaching resistance, and improve fluorescence Effect of Luminous Intensity

Inactive Publication Date: 2013-03-06
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

The nano-fluorescent biological probes that have been developed mainly include: (1) semiconductor nanocrystals (also known as quantum dots); (2) composite nano-fluorescent particles with fluorescent molecules inside (one type is polymer plastic matrix nano-fluorescent particles, One type is nano silica gel particles containing various fluorescent substances) (document 10: W.H.Liu, M.Howarth, A.B.Greytak, Y.Zheng, Nocera, D.G.A.Y.Ting, M.G.Bawendi, J.Am.Chem.Soc.2008, 130 , 1274; Literature 11: X.J.Zhao, L.R.Hilliard, S.J.Mechery, Y.P.Wang, R.P.Bagwe, S.G.Jin, W.H.Tan, Proc.Natl.Acad.Sci.USA2004, 101, 15027), but these fluorescent particles are used for bioassay However, there are still problems such as luminescent flickering, complicated labeling process, easy leakage of fluorescent molecules, and serious background fluorescence interference.
[0005] Recently, several composite nano-fluorescent particles based on europium and terbium complexes have been developed and used for high-sensitivity time-resolved fluorescence immunoassay and imaging detection (Document 12: Y.Xu, Q.G.Li, Clin.Chem.2007, 53, 1503; Literature 13: P. Huhtinen, M. O. Kuronen, V. Hagren, H. Takalo, H. Tenhu, T. , H. , Anal.Chem.2005, 77, 2643; Literature 14: Yuan Jingli, Tan Minggan, Ye Zhiqiang, Wang Guilan, A functional nano-rare earth fluorescent particle and its preparation and application, Chinese invention patent, patent number: ZL02144517.6; Literature 15: Yuan Jingli, Tan Mingqian, Hai Xiaodan, Wang Guilan, β-diketone-trivalent europium complex nanofluorescent probe and its preparation and application, Chinese invention patent, patent number: ZL03133857.7; Document 16: J.Wu, G.L.Wang , D.Y.Jin, J.L.Yuan, Y.F.Guan, J.Piper, Chem.Commun.2008, 365), however, these nano-rare earth fluorescent particles still cannot avoid the use of high-energy ultraviolet excitation light sources, so they are limited in some application fields

Method used

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  • Nanometer europium fluorescent particle with performance of visible light excitation, preparation method and application thereof
  • Nanometer europium fluorescent particle with performance of visible light excitation, preparation method and application thereof
  • Nanometer europium fluorescent particle with performance of visible light excitation, preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0038] Example 1: Preparation and Characterization of Nano Europium Fluorescent Particles Excited by Visible Light

[0039] (1) Fluorescent precursor APS-BHHCT-Eu 3+ - Preparation of DPBT

[0040] Dissolve 25 μmol of BHHCT in 4 mL of anhydrous tetrahydrofuran, add 62.5 μmol of aminopropyltriethoxysilane (APS) under stirring, stir at room temperature for 3 hours, then add 15 mL of anhydrous tetrahydrofuran solution dissolved in 50 μmol of DPBT, and add 25 μmol EuCl 3 ·6H 2 O, stirred for 24 hours, evaporated the solvent to dryness with a rotary evaporator and dried in vacuum to obtain the fluorescent precursor APS-BHHCT-Eu 3+ -DPBT.

[0041] (2) Preparation of nano-europium fluorescent bioprobes excited by visible light

[0042] The above fluorescent precursor APS-BHHCT-Eu 3+ - DPBT (2 mg, 4 mg, 6 mg, 8 mg and 10 mg) was dissolved in 1.5 mL of toluene and added to a solution containing 7.25 g of cyclohexane, 2.37 g of Triton X-100, 1.82 g of n-octanol and 0.55 mL of water...

Embodiment 2

[0053] Example 2: Nano europium fluorescent particles are used as markers for time-resolved fluorescent immunoassay for human prostate-specific antigen (PSA for short)

[0054] (1) Nano europium fluorescent particles labeled streptavidin (SA for short)

[0055] Add 1.0 mg of nano-europium fluorescent particles and 3.0 mg of bovine serum albumin (BSA) with 0.1 mol L of pH 7.1 -1 After 1.2 mL of phosphate buffer solution was completely dissolved, 0.3 mL of 1% glutaraldehyde aqueous solution was added under stirring, and stirred at room temperature for 24 hours. After the nanoparticles were collected by centrifugation and fully washed with phosphate buffer solution, 0.9 mL of phosphate buffer solution containing 0.2 mg of SA and 0.2 mL of 1% glutaraldehyde aqueous solution were added, stirred at 4 °C for 22 hours, and 2.0 mg of sodium borohydride was added to continue the reaction 2 Hour. The nanoparticles marked with SA molecules on the surface were centrifuged and fully washe...

Embodiment 3

[0062] Example 3: Nano-europium fluorescent particles are used as markers for time-resolved fluorescence microscopy imaging to determine pathogenic microorganisms in complex environmental samples-Giardia lamblia (Giardia lamblia)

[0063] (1) Time-resolved fluorescence microscopy imaging of Giardia lamblia in complex environmental samples Take 5 μL of Giardia lamblia (2×10 6 1 / mL) was added to 15 μL of 10000-fold concentrated environmental water samples containing a large number of various impurities, and then 16 μL of anti-Giardia lamblia monoclonal antibody (50 μg / mL), 16 μL of biotin-labeled rabbit anti-mouse II Secondary antibody (50 μg / mL, prepared in the same way as biotin-labeled goat anti-human PSA antibody) and 6 μL nanofluorescent particle-labeled SA solution, incubated at room temperature for 24 hours, low-speed centrifugation (500 rpm) to collect Giardia lamblia and Redisperse in water several times to remove unreacted nanoparticles and antibodies. The Giardia lam...

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Abstract

The invention discloses a preparation method of strong fluorescence nanometer europium fluorescent particles with performance of visible light excitation and the application thereof in biological detection technology thereof. Firstly, an amino silicone alkylating agent is utilized for forming a fluorescent precursor with a ternary europium complex excited by a visible light, and then the fluorescent precursor and silicon ester are polymerized in a water-in-oil type microemulsion to generate the nanometer europium fluorescent particles with the surface provided with active groups. The nanometer europium fluorescent particles have strong visible excitation peaks, the fluorescent lifetime is about 390 microseconds, and the fluorescence quantum yield can reach 66%. The influence of the short-lived background fluorescence can be effectively eliminated when the nanometer europium fluorescent particles are used as biological fluorescent markers for time-resolved fluorescence bioassay, the light stability of the markers can be remarkably enhanced, and the light injury to biological samples is reduced by adopting visible light excitation, and the invention has important application prospect in the fields of chemical and biological detection of time-resolved fluorescence immunoassay, living cell, biological staining and time-resolved fluorescence microscope image analysis, and the like.

Description

technical field [0001] The invention relates to a visible light-excited nano europium fluorescent biomarker material, in particular to the preparation of nano europium fluorescent particles with visible light excitation properties that can be used for biomolecular labeling and its application in time-resolved fluorescent biodetection. Background technique [0002] Rare earth (europium and terbium) fluorescent complexes have very special fluorescence properties, such as long fluorescence lifetime, large Stokes shift and sharp emission spectrum. Based on these characteristics, the time-resolved fluorescent immunoassay technology using rare earth fluorescent complexes as markers has made significant progress in the past 20 years, and is playing an increasingly important role in the practice and research of medicine and life sciences. In recent years, the time-resolved fluorescent bioimaging analysis technology based on rare earth fluorescent complexes has also become a useful t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09K11/06C08G77/26C08G77/28G01N21/64
Inventor 吴晶袁景利关亚风
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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