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Double labelling Nano-Au probe and preparation method

A nano-gold probe and nano-gold technology, applied in the double-labeled nano-gold probe and its preparation, the application field of the nano-gold probe in the detection of ultra-trace antigens, which can solve the problems of inability to use immunoassays and difficulties in practical promotion and application, etc. problems, to achieve good clinical application value, improve detection sensitivity, and easy operation methods

Inactive Publication Date: 2014-07-16
SHENZHEN PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] Invention Patent Application Publication (Publication No.: CN1339609A; Country: China; Publication Date: March 13, 2002) discloses a nano-gold labeled gene probe, which is formed by combining a nano-gold with an alkylthiol-modified nucleic acid. Probes are used in the field of diagnostic gene chips, but they cannot be used in immunoassays, and there are difficulties in their practical application

Method used

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  • Double labelling Nano-Au probe and preparation method
  • Double labelling Nano-Au probe and preparation method
  • Double labelling Nano-Au probe and preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Trisodium citrate (Shanghai Sinopharm) reduced HAuCl 4 Preparation of colloidal gold. 100ml of 0.01% HAuCl 4 Solution is heated to boiling; Under high-speed stirring condition (1200rpm), add 5ml 1% trisodium citrate (Na 3 C 6 h 5 o 7 2H 2 0), continue heating and stirring for 30min, cool naturally, and return to the original volume with ultrapure water. Filter through a 0.22 μm filter membrane and store in the dark at 4°C for later use.

[0061] Such as figure 1 As shown, the resulting nano-gold was observed with a transmission electron microscope (TEM) to observe the morphology of the particles, and its size and particle size distribution were measured with a laser scattering instrument. concentration of gold nanoparticles.

[0062] Experimental results The obtained nano-gold has the largest absorption peak at 518nm, uniform particle size, good dispersion, the main distribution range is 10±3nm, and the approximate concentration of gold colloid is 3.7nM.

[0...

Embodiment 2

[0065]Get 100ml 0.01% HAuCl solution and heat it to boiling; add 2.5-4.5ml 1% trisodium citrate (Na 3 C 6 h 5 o 7 2H 2 0), continue heating and stirring for 30min, cool naturally, and return to the original volume with ultrapure water. Filter through a 0.22 μm filter membrane and store in the dark at 4°C for later use.

[0066] Experimental results The particle size of the obtained nano gold particles has a maximum absorption peak at about 520-534nm, and the particle size is about 15-30nm.

[0067] 2. Determination of the optimum pH and optimum stable amount of antibody-bound gold nanoparticles

Embodiment 3

[0069] Adopt the nano-gold prepared in Example 1 of optimized preparation conditions, get several 1.5ml small test tubes, add 1ml nano-gold solution respectively; 2 CO 3 Adjust the pH to 3, 4, 5, 6, 7, 8, 9, and 10 in turn; take a 96-well plate, add 100 μl of the above colloidal gold into the wells according to the pH from low to high, and repeat; Add 2μl of antigen or antibody with a concentration of 1mg / ml to the wells, mix, and place at room temperature for 10-15min; then add a certain amount of 1.2M NaCl solution to each well, mix, and place at room temperature for 10min; Observe the color change of colloidal gold , record the lowest pH (X) that keeps red; and repeat the above steps, the pH gradient is X-0.6; X-0.3; X; X+0.3; X+0.6; X+1. Observe the color change of the colloidal gold until it is placed at room temperature for 2 hours, and record the lowest pH that remains red.

[0070] The experimental results show that the optimal pH for the combination of the gold nano...

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Abstract

The invention discloses a double-labeled nano-gold probe, its preparation method and application. The probe includes: the gold nanoparticle is the core, and the surface is simultaneously connected with antibodies and oligonucleotides. The preparation method of the double-labeled nano-gold probe includes the following steps: 1. Ligation reaction: add a concentration of oligonucleotide solution and antibody solution to the nano-gold sol, mix evenly, and make the nano-gold particles and antibody in the solution 2. Nucleic acid can fully function to make a mixed solution; ② Stabilization and blocking reaction: prepare a PBS buffer solution containing 5-10% of SDS, then add it to the mixed solution made in step ①, add a blocking agent, and form a nano Sol of the probe. The double-labeled nano-gold probe can transform the detection of antigens into the detection of nucleic acid substances, and is applied to the detection of tumor markers, hormones, and trace proteins of pathogens.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a double-labeled nano-gold probe with biological activity, a preparation method thereof and the application of the nano-gold probe in ultra-trace antigen detection. Background technique [0002] In recent years, medical diagnostic technology has developed rapidly, and immunoassay technology has also changed with each passing day. As an important branch of immunoassay technology, marker immunoassay technology is the most active. [0003] In 1959, American scholars Berson and Yalow established the radio immunoassay (Radio Immunoassay, RIA) method, which opened up a new field of marker immunoassay. Because RIA has the advantages of high sensitivity, strong specificity, and simplicity, RIA has certain vitality, but because it must use radionuclides, it needs protection and pollution prevention; the effective use time of markers is short, and it is difficult to realize au...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/52G01N33/53G01N33/577
Inventor 李富荣齐晖任莉莉孔小丽周汉新
Owner SHENZHEN PEOPLES HOSPITAL
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