CN-121978095-A - Mycobacterium tuberculosis detection probe and preparation method and application thereof

CN121978095ACN 121978095 ACN121978095 ACN 121978095ACN-121978095-A

Abstract

The invention belongs to the technical field of biomedical detection and immunoassay, and particularly discloses a mycobacterium tuberculosis detection probe and a preparation method and application thereof. The mycobacterium tuberculosis detection probe provided by the invention comprises a capture probe and a signal probe, wherein the capture probe is a magnetic nanoparticle with a mycobacterium tuberculosis specific antibody 1 immobilized on the surface, and the signal probe is a mycobacterium tuberculosis specific antibody 2 with a photosensitizer marked on the surface. The mycobacterium tuberculosis detection probe can realize high-sensitivity detection of mycobacterium tuberculosis under mild conditions by combining a photosensitizer marking technology with immunoassay, can catalyze a plurality of chromogenic substrates to perform oxidation reaction under visible light irradiation to realize rapid colorimetric detection of the mycobacterium tuberculosis, is particularly suitable for clinical serum sample detection and other requirements, and has the advantages of simple preparation process, strong recognition specificity, high detection sensitivity, good stability and reproducibility and the like.

Inventors

LAN MINHUAN
LEI TINGTING
Zhao Shaojing
YI JIANING
ZENG JIE
LIU LUYAO
YU JIE

Assignees

中南大学

Dates

Publication Date: 20260505
Application Date: 20260207

Claims (10)

1. A mycobacterium tuberculosis detection probe is characterized by comprising a capture probe and a signal probe, wherein the capture probe is a magnetic nanoparticle with a mycobacterium tuberculosis specific antibody 1 immobilized on the surface, and the signal probe is a mycobacterium tuberculosis specific antibody 2 with a photosensitizer marked on the surface.
2. The Mycobacterium tuberculosis detection probe according to claim 1, wherein the specific antibody 1 and the specific antibody 2 are specific antibodies against different antigens of Mycobacterium tuberculosis, wherein the antigens comprise CFP10 protein, ESAT6 protein, MPT64 protein, 38kD protein, ag85B protein, MTC28 protein or MoeX protein of Mycobacterium tuberculosis.
3. The mycobacterium tuberculosis detection probe according to claim 1, wherein the mycobacterium tuberculosis specific antibody 1 is an anti-CFP 10 protein monoclonal antibody, and the mycobacterium tuberculosis specific antibody 2 is an anti-ESAT 6 protein monoclonal antibody, an anti-Ag 85B protein monoclonal antibody or an anti-MTC 28 protein monoclonal antibody.
4. The mycobacterium tuberculosis detection probe according to claim 1, wherein the surface of the magnetic nanoparticle is modified with a functional group, the functional group comprises at least one of carboxyl, amino or streptavidin, and the magnetic nanoparticle is selected from at least one of magnetic ferroferric oxide nanoparticle, nickel magnetic nanoparticle or cobalt magnetic nanoparticle; The photosensitizer is at least one selected from phthalocyanine compounds, porphyrin compounds, phenothiazine dyes, xanthene dyes, flavin compounds or natural photosensitizers.
5. The method for preparing a Mycobacterium tuberculosis detection probe according to any one of claims 1 to 4, comprising the steps of: s1, mixing magnetic nano particles with a mycobacterium tuberculosis specific antibody 1 in a buffer solution in the presence of a cross-linking agent for reaction, collecting a product through magnetic separation, and sealing to obtain a capture probe; s2, mixing the mycobacterium tuberculosis specific antibody 2 with a photosensitizer in a buffer solution in the presence of a coupling agent for reaction, and purifying to obtain a signal probe; In the step S2, the molar ratio of the mycobacterium tuberculosis specific antibody 2 to the photosensitizer is 1 (5-7).
6. The preparation method according to claim 5, wherein in the step S1, the cross-linking agent comprises at least one of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, bis (sulfosuccinimidyl) suberate, disuccinimidyl glutarate and 3,3' -dithiobis (sulfosuccinimidyl propionate), the buffer solution comprises at least one of phosphate buffer solution, tris-HCl buffer solution, HEPES buffer solution, MES buffer solution and MOPS buffer solution, the pH value of the buffer solution is 6.0-8.5, the temperature of the mixing reaction is 15-30 ℃ and the time is 1-3 h.
7. The preparation method according to claim 5, wherein in the step S2, the coupling agent comprises at least one of N-gamma-maleimidobutyloxy succinimidyl ester, sulfo-succinimidyl-6- (3' - [ 2-pyridyldithio ] -propionamido) caproate or 4- (N-maleimidomethyl) cyclohexane-1-carboxylic sulfosuccinimidyl ester sodium salt, the buffer comprises at least one of carbonate buffer, phosphate buffer and borate buffer, the pH value of the buffer is 6.5-9.0, the mixing reaction is carried out under a light-shielding condition, the reaction temperature is 2-8 ℃ and the time is 2-5 h, and the purification method comprises at least one of dialysis, gel filtration chromatography and ultrafiltration centrifugation.
8. The use of a mycobacterium tuberculosis detection probe according to any one of claims 1-4 for detecting mycobacterium tuberculosis in a sample, wherein the method for detecting mycobacterium tuberculosis in a sample comprises the steps of: S10, performing immune reaction, namely mixing and incubating a capture probe with a sample solution to combine mycobacterium tuberculosis with a specific antibody 1, and adding a signal probe to form a sandwich immune complex; s20, separating and washing, namely performing magnetic separation, washing and removing unbound substances; S30, photosensitive color development, namely adding a color development substrate into the washed compound, and carrying out photosensitive oxidation reaction under visible light irradiation; s40, detecting and analyzing, namely measuring the absorbance value of a reaction system, and calculating the concentration of the mycobacterium tuberculosis in the sample to be detected according to a standard curve; The chromogenic substrate comprises at least one of 3,3', 5' -Tetramethylbenzidine (TMB), 2 '-biazabis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS), o-phenylenediamine (OPD), 3-amino-9-ethylcarbazole (AEC) or 3,3' -Diaminobenzidine (DAB).
9. The method according to claim 8, wherein in step S10, the incubation is performed at 20-45 ℃ for 15-50 min, the buffer is phosphate buffer, HEPES buffer or Tris-HCl buffer, and the pH of the buffer is 6.0-8.5.
10. A mycobacterium tuberculosis detection kit comprising the mycobacterium tuberculosis detection probe of any one of claims 1-4.

Description

Mycobacterium tuberculosis detection probe and preparation method and application thereof Technical Field The invention relates to the technical field of biomedical detection and immunoassay, in particular to a mycobacterium tuberculosis detection probe and a preparation method and application thereof. Background Mycobacterium tuberculosis (Mycobacterium tuberculosis, mtb) is the causative agent of tuberculosis, which is one of the major infectious diseases that lead to death worldwide. Mycobacterium tuberculosis infection can cause various diseases such as pulmonary tuberculosis, lymphoid tuberculosis, tuberculous pleurisy and the like, and seriously threatens human health. Therefore, the method can rapidly and accurately detect the mycobacterium tuberculosis antigen in clinical samples (such as sputum, pleural effusion, tissue fluid and the like), and has important clinical significance for early diagnosis, differential diagnosis, treatment scheme formulation and curative effect monitoring of tuberculosis. Currently, the mainstream methods for detecting Mtb in clinical laboratories are mainly based on immunoassay principles, including enzyme-linked immunosorbent assay, chemiluminescent immunoassay, electrochemiluminescent immunoassay, and the like. These methods typically rely on enzyme-labeled (e.g., horseradish peroxidase, HRP) or chemiluminescent-labeled secondary antibodies to effect detection by catalyzing the production of color, light, or electrical signals from the substrate. Although these techniques are well established, there are some inherent limitations in that, firstly, the activity of the enzyme labels is susceptible to environmental temperature, pH and storage conditions, stability is relatively poor, possibly resulting in batch-to-batch differences, and secondly, the chemiluminescent method, while having high sensitivity, generally requires expensive instruments and special reagents, has high detection cost, and in addition, some methods have complicated operation steps, have long detection time, and are difficult to meet the requirements of rapid detection in clinical emergency or surgery. In recent years, researchers have begun to explore non-enzyme dependent signal amplification strategies in an effort to find more stable, convenient detection schemes. Wherein, the nanometer material which simulates enzyme catalysis, in particular has peroxidase activity, has shown good application prospect. However, there is still room for improvement in the catalytic efficiency, stability and biocompatibility of these mimic enzymes. The photosensitizing oxidation reaction is an emerging signal generation mechanism, which utilizes a photosensitizer to generate active oxygen (such as singlet oxygen, hydroxyl radicals and superoxide anions) under specific wavelength illumination, and further oxidizes a colorless chromogenic substrate (such as TMB) to generate color change. The process does not depend on biological enzyme, and has the potential advantages of mild reaction conditions, easiness in control, stable signals and the like. If the photosensitive oxidation reaction and the high-specificity immune recognition reaction can be combined, a novel Mtb detection probe is constructed, partial defects of the traditional enzyme labeling technology are hopeful to be overcome, and a new Mtb detection method with better performance is provided for clinic. Therefore, there is a need to develop a new method for Mtb immunodetection with high sensitivity, high specificity, good stability and simple operation. Disclosure of Invention The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a Mycobacterium tuberculosis (Mtb) detection probe based on a photo-sensitive oxidation reaction. The Mtb detection probe can realize high-sensitivity detection of Mtb under mild conditions by combining a photosensitizer marking technology with immunoassay, can catalyze a plurality of chromogenic substrates to perform oxidation reaction under visible light irradiation, realizes quick colorimetric detection of Mtb, is particularly suitable for medical detection requirements such as clinical serum sample detection, disease diagnosis screening and treatment effect monitoring, and has the advantages of simple preparation process, strong recognition specificity, high detection sensitivity, good stability and reproducibility and the like. The invention also provides a preparation method of the detection probe. The invention also provides application of the detection probe. In a first aspect of the invention, a mycobacterium tuberculosis detection probe is provided, the detection probe comprises a capture probe and a signal probe, the capture probe is a magnetic nanoparticle with a mycobacterium tuberculosis specific antibody 1 immobilized on the surface, and the signal probe is a mycobacterium tuberculosis specific antibody 2 with a photosensitize