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Method for quantitative detection of ultramicro-protein based on principle of biomineralization

A biomineralization and protein technology, which is applied in the field of ultra-trace protein quantitative detection, can solve the problems of limited recognition ability of specific target proteins, reduced detection sensitivity and specificity, and sensitivity to the surrounding environment, and achieves improved selectivity and sensitivity. The effect of stable properties and simple preparation process

Inactive Publication Date: 2011-11-02
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the existing surface-unmodified gold nanoparticles and the protein to be tested rely on electrostatic or surface tension interactions, are coupled in a non-covalent manner, are easy to dissociate, often have adhesion and agglomeration phenomena, and are sensitive to the surrounding environment. , reducing the sensitivity and specificity of detection
Moreover, the existing protein quantification technology has limited ability to identify specific target proteins, low detection sensitivity, poor stability, long detection cycle, and high cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0025] With dendrimers (PPIHA) and chloroauric acid (HAuCl 4 ) as raw material, the surface aminated nano gold seed sol was prepared by microwave reflux heating method. Dissolve 1.5 mL of 0.67% chloroauric acid solution in 100 mL of ultrapure water, add 90 μL of 5% PPIHA in toluene; stir and mix well, then heat with microwave under reflux for 5 min.

[0026] Add 180 μL of 1% bovine serum albumin (Bovineserum albumin, BSA) solution to 4mL of the prepared nano-gold seed sol, then add 120 μL of 1% hydrochloric acid, dilute to 6ml with ultrapure water after shaking up, and react to generate AuNPs- BSA conjugate.

[0027] Mix 1.8mL of AuNPs-BSA conjugate with 0.6mL of phosphate buffer at pH 7.0, shake well, dilute to 10mL with ultrapure water, and set aside. A certain amount (in the range of 0-180 μL) of the above-mentioned AuNPs-BSA conjugate was taken at intervals of 10 μL, and added to 3.00 mL of ultrapure water. Then add 2mL 0.02% chloroauric acid solution, 2mL sodium citrat...

Embodiment approach 2

[0031] Dendrimers (PPIHA) and chloroauric acid (HAuCl 4 ) as raw material, the surface aminated nano gold seed sol was prepared by microwave reflux heating method. Dissolve 1.2mL of 0.67% chloroauric acid solution in 100mL of ultrapure water, add 85μL of 5% PPIHA in toluene; stir and mix well, then heat with microwave for 5min.

[0032]Add 180 μL, 1% bovine serum albumin (Bovine serum albumin, BSA) solution to 4.5mL of the nano-gold seed sol prepared in step (1), add 115 μL of 1% hydrochloric acid, dilute with ultrapure water to 6ml to prepare the AuNPs-BSA conjugate.

[0033] Mix 1.5mL AuNPs-BSA conjugate with 0.6mL pH 7.0 phosphate buffer, shake well, dilute to 10mL with ultrapure water, and set aside. A certain amount (in the range of 0-180 μL) of the above-mentioned AuNPs-BSA conjugate was taken at intervals of 10 μL, and added to 3.00 mL of ultrapure water. Then add 1.6mL 0.02% chloroauric acid solution, 2mL sodium citrate-phosphate buffer solution with pH 2.26 and 2.5...

Embodiment approach 3

[0037] With dendrimers (PPIHA) and chloroauric acid (HAuCl 4 ) as raw material, the surface aminated nano gold seed sol was prepared by microwave reflux heating method. Dissolve 2 mL of 0.67% chloroauric acid solution in 135 mL of ultrapure water, add 95 μL of 5% PPIHA in toluene; stir and mix well, then heat under microwave reflux for 5 min.

[0038] Add 200 μL of 1% bovine serum albumin (Bovineserum albumin, BSA) solution to 5mL of the prepared nano-gold seed sol, then add 130 μL of 1% hydrochloric acid, shake up and dilute to 6ml with ultrapure water, and the reaction generates AuNPs- BSA conjugate.

[0039] Mix 2mL of AuNPs-BSA conjugate with 0.8mL of pH 7.0 phosphate buffer, shake well, dilute to 12mL with ultrapure water, and set aside. A certain amount (in the range of 0-180 μL) of the above-mentioned AuNPs-BSA conjugate was taken at intervals of 15 μL, and added to 3.00 mL of ultrapure water. Then add 2mL 0.02% chloroauric acid solution, 2.8mL pH 2.26 sodium citrate...

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Abstract

The invention discloses a method for quantitative detection of ultramicro-protein based on the principle of nano gold particle biomineralization, which comprises the following steps: firstly, by taking the first generation of PPIHA dendrimer and chloroauric acid (HAuCl4) as raw materials, preparing nano gold colloid with surface modified by amine by means of a microwave reflux heating method, andcombining the nano gold colloid with to-be-detected protein to form conjugate at a normal temperature; secondly, carrying out biomineralization on the nano gold-protein conjugate in a sodium citrate-chloroauric acid system; and then carrying out resonance scattering spectral analysis on the biomineralization product by using a fluorescence spectrum spectrograph. Because of the linear relation between the peak strength of the resonance scattering spectrum of the product at the characteristic wavelength and the protein concentration, the ultramicro-protein can be quantitatively detected. The method is easy and quick and has high sensitivity, high specificity and high accuracy. In addition, the method has a good portability and can be used for early tumor diagnosis, quick food safety detection, water quality monitoring and the like.

Description

technical field [0001] The invention relates to an ultra-trace protein quantitative detection method based on the principle of biomineralization. Background technique [0002] In recent years, noble metal nanoparticles have been widely used in biological detection and analysis technology, which has played an important role in promoting the development of life sciences. With precious metal nanoparticles as the core, a series of new biomolecular analysis and detection technologies have been developed, which play an important role in various research fields related to biomedicine, especially ultra-trace molecular recognition, early diagnosis of tumors, rapid detection of food safety, water quality monitoring, etc. It has had a profound impact on the development of ultra-trace biomolecular analysis technology. [0003] Among them, gold nanoparticles (AuNPs) have been widely used in the design of bio-optical probes due to their scale-dependent physicochemical properties. The de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/66B82Y15/00
Inventor 刘钊李剑君朱键赵军武
Owner XI AN JIAOTONG UNIV
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