HPRT (hypoxanthine phosphoribosyl transferase) body gene mutation detection method based on surface plasmon resonance

A surface plasmon and detection method technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, and measuring devices, etc., to achieve the effects of simple operation, high sensitivity, and high data accuracy

Inactive Publication Date: 2011-08-24
NANJING UNIV OF AERONAUTICS & ASTRONAUTICS
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

At present, there is no literature report using SPR-based technology to detect HPRT somatic gene mutations. The present invention provides a method for detecting HPRT somatic gene mutations based on SPR technology for the first time

Method used

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  • HPRT (hypoxanthine phosphoribosyl transferase) body gene mutation detection method based on surface plasmon resonance
  • HPRT (hypoxanthine phosphoribosyl transferase) body gene mutation detection method based on surface plasmon resonance
  • HPRT (hypoxanthine phosphoribosyl transferase) body gene mutation detection method based on surface plasmon resonance

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Embodiment １

[0028] Based on surface plasmon resonance technology 60 Co gamma ray irradiation-induced HPRT somatic gene mutation detection method:

[0029] Part 1: SPR method to detect wild-type HPRT standard

[0030] 1. Preparation of 6-thioguanine-sensitive SPR gold membrane: 20 ul of cysteine ​​(20 mM) was covered in Pirrahan solution (H 2 o 2 : Concentrated sulfuric acid = 3: 1) Wash the surface of the gold film (cleaning time is 15s), and then place the gold film in the dark overnight; after overnight, the gold film is thoroughly washed with distilled water to remove unbound cysteine, nitrogen Blow dry; the surface of the cysteine-modified gold film was covered with carboxymethyl dextran, N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) A mixture of carbodiimide hydrochloride (EDC) (15 mM NHS, 75 mM EDC and 10 mg / ml carboxymethyl dextran) was placed in the dark for 2.5 hours. After 2.5 hours, rinse with distilled water and dry with nitrogen.

[0031] 2. Fix the ca...

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Abstract

The invention belongs to a quantitative detection method of an enzyme, and in particular relates to an HPRT (hypoxanthine phosphoribosyl transferase) body gene mutation detection method based on surface plasmon resonance. The HPRT body gene mutation detection method based on surface plasmon resonance is characterized by comprising the following steps: (1) preparation of a wild HPRT enzyme-sensitive SPR (surface plasmon resonance) gold film; (2) SPR detection of a wild HPRT standard substance; and (3) sample preparation and the SPR detection: after collected mammal cells are repeatedly washed by a hanks liquid, directly adding a histiocyte lysis solution; vibrating at the temperature of 4DEG C to cause the sample to fully crack; carrying out low-temperature centrifugation on the cracked product; collecting a supernate containing proteins; replacing the lysis solution component in the supernate by PBS (phosphate buffered saline), and storing in a refrigerator at the temperature of 4 DEG C; and carrying out SPR detection. The method has high detection sensitivity, and the content of wild HPRT of ngmL-1 order of magnitude can be detected.

Description

technical field [0001] The invention belongs to a method for quantitatively detecting enzymes, in particular to a method for detecting mutations in HPRT body genes based on surface plasmon resonance. Background technique [0002] Epidemiological surveys and experimental studies have shown that environmental physical and chemical factors are the main cause of tumor onset, and the coincidence rate of mutagens and carcinogens is 80%. Therefore, it is of great significance to establish appropriate methods to detect mutagenesis of physicochemical factors to reduce the risk of carcinogenesis. Mammalian cell hypoxanthine-guanine phosphoribosyl transferase (Hypoxanthine-Guanine Phosphoribosyl Transferase, HPRT) is a cytoplasmic enzyme. Studies at home and abroad have shown that its gene loci are more sensitive to chemical mutagens and ionizing radiation. Based on this feature, the in vitro HPRT gene mutation detection method in mammalian cells is used to evaluate the damage caused...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/63G01N33/573G01N1/28C12Q1/48
Inventor 张晓红张海黔
Owner NANJING UNIV OF AERONAUTICS & ASTRONAUTICS
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