Double labelling Nano-Au probe and preparation method and application thereof

A nano-gold probe and double-labeled technology, which is applied in material testing products, measuring devices, instruments, etc., can solve the problems of practical application difficulties, inability to use immunoassays, etc., to achieve good clinical application value, improve detection sensitivity, and operation. The effect of the simple method

Inactive Publication Date: 2009-10-07
SHENZHEN PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0011] Invention Patent Application Publication (publication number: CN 1339609A; Country: China; Publication Date: March 13, 2002) discloses a nano-gold labeled gene probe, in which a nano-gold is used to combine with a nucleic acid modified by an alkylthiol group. Probes are formed and used in the field of diagnostic gene chips, but they cannot be used in immunoassays, and there are difficulties in practical promotion and application

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0058] 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.

[0059] like figure 1 As shown, the resulting nano-gold is observed with a transmission electron microscope (TEM) for particle morphology, its size and particle size distribution are measured with a laser scattering instrument, and the absorption peak is scanned with a UV-visible spectrophotometer and estimated according to the Lambert-Beer law concentration of gold nanoparticles.

[0060] Experimental results The obtained nano-gold has the largest absorption peak at 518nm, uniform particle size, good dispersion, the main distr...

Embodiment 2

[0063] 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 2H2 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.

[0064] 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.

[0065] 2. Determination of the optimum pH and optimum stable amount of antibody-bound gold nanoparticles

Embodiment 3

[0067] 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.

[0068] 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 labelling Nano-Au probe and a preparation method and application thereof. The probe comprises a Nano-Au particle as a core, the surface of which is connected with antibody and oligonucleotide simultaneously. The preparation method of the double labelling Nano-Au probe includes the following steps: (1) coupled reaction: adding oligonucleotide solution and antibody solution in Nano-Au sol with certain concentration and uniformly mixing the solutions to lead the Nano-Au particle, the antibody and nucleic acid to fully act in the solution; and (2) stabilizing and blocking reaction: preparing PBS buffer solution with the content of SDS being 5-10 percent and then adding the PBS buffer solution in the mixed solution prepared in step (1) and adding sealant to form nano probe sol. The double labelling Nano-Au probe can convert the antigenic detection into the detection of nucleic acid matter and is applied to the detection of tumor marker, hormone and pathogene micro-protein.

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 Applications(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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