Homogeneous creatine kinase chemiluminiscence detection reagent and preparation method thereof

Abstract

The invention discloses a homogeneous creatine kinase chemiluminiscence detection reagent and a preparation method thereof. The method comprises steps of adopting gold nanoparticles, adding a water-soluble photosensitizer into the gold nanoparticles and acquiring a nanogold-photosensitizer compound, coupling the nanogold-photosensitizer compound with a creatine kinase monoclonal antibody, and generating electrostatic adsorption between the two under the isoelectric point environment condition of the creatine kinase monoclonal antibody through the charge property of the nanogold particles, so that the creatine kinase monoclonal antibody is coupled to the surfaces of the nanogold particles. The homogeneous creatine kinase chemiluminiscence detection reagent is obtained according to the preparation method disclosed by the invention, the utilization rate of the photosensitizer is increased, the production process is simplified, the production cost is reduced, a sandwich method detection principle is adopted to detect a detected object in use, the effective light dose of irradiation is increased, more photosensitizers are activated, a stronger photosensitive effect is generated, the difference between the excitation light wavelength and the emission wavelength is increased, and the detection identification degree is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a homogeneous creatine kinase chemiluminescent detection reagent and a preparation method. Background technique [0002] Creatine kinase CK is a large dimeric protein, in which the M subunit has a small N-terminal structure and a large C-terminal structure; the M-type subunit consists of 387 amino acid residues and has a molecular weight of about 43kDa , There are 8 sulfhydryl groups in the molecule, but no disulfide bonds. Natural creatine kinase is a compact spherical structure. Recent studies on the relationship between conformational changes and activity changes of creatine kinase have shown the flexibility of the conformation of the active site of the enzyme molecule, that is, the conformation of the microdomain of the active site of the enzyme molecule is easily affected by denaturants. Changes lead to rapid inactivation of enzyme molecules, and at this time the overall confor...

AI Technical Summary

Problems solved by technology

[0003] For the detection method of creatine kinase, the existing light-induced chemiluminescence method needs to use non-water-soluble photosensitizers (porphyrin, chlorophyll, phthalocyanine and their derivatives, etc.), and the singlet oxygen quantum yield of unmodified photosensitizers Low, affecting detection performance; and derivatized photosensitizer, chemical synthesis is difficult, complex process

[0004] The photosensitizer used in the existing photoluminescence system needs to be embedded in polystyrene microspheres in the organic phase, which needs to be purified by complex centrifugation, ultrafiltration, etc., and needs to be restored to the aqueous matrix. It is easy to produce loss during the process of burying and purification, which reduces the photosensitive effect

[0005] Existing light-induced chemiluminescence technology has little difference between the excitation wavelength and the detection wavelength, and is susceptible to interference, and the fluorescence background is greatly affected

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