Multidimensional tumor detection combination reagent based on in-situ intracellular mineralization and application thereof

Abstract

The application discloses a multi-dimensional tumor detection reagent based on in-situ intracellular mineralization and application thereof, which comprises reagent 1 and reagent 2; the reagent 1 is chloroauric acid; and the reagent 2 is ultrapure water containing sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate. The technical scheme of the application is different from the prior art in that gold nanostructures are synthesized in vitro, and then tumor cells are identified through other specific compound molecules. In the application, gold nanostructures are synthesized through liquid-liquid phase separation in tumor cells, have good specificity, and do not need the step of in-vitro synthesis, thus being convenient and simple. Secondly, the application has the advantages of convenient and fast synthesis and low cost, and does not need to add other dangerous chemicals. Thirdly, the application can realize near-infrared fluorescence labeling of tumor tissues / cells and molecular recognition and detection of surface-enhanced Raman spectroscopy through simple operation. Finally, the application has high repeatability and can realize analysis, identification and imaging of chemical molecules of tumor cells at a single cell level.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor detection, and specifically relates to a combination reagent for multidimensional tumor detection based on in situ intracellular mineralization and its application. Background Art

[0002] The current surface-enhanced Raman spectroscopy (SERS) method for detecting tumors involves synthesizing nanoparticles or nanosheets with SERS properties in vitro, then co-incubating them with tumor tissue. After active uptake of the nanoparticles by tumor cells or normal cells, Raman spectroscopy is performed for detection and classification. This in vitro synthesis of nanoparticles and their introduction into cells is complex, with uncontrollable surface functional groups. Introduction into cells can lead to uneven distribution within the cell, with the particles typically being taken up into lysosomes via endocytosis. The acidic environment and uneven distribution prevent stable and reliable Raman spectral information from being obtained, ultimately leading to failure in tumor classification and detection. Furthermore, it is difficult to analyze and identify ex vivo tumor tissue.

[0003] On the other hand, the same is true for fluorescent probes for tumor detection. It is necessary to introduce the tumor in vitro for imaging detection, and the results are consistent with the above situation. Alternatively, gold ions are introduced into the mitochondria of the cells to synthesize gold nanoclusters in situ through ROS to achieve fluorescence imaging of the tumor. This fluorescence detection produced by synthesizing gold nanoclusters through ROS in the mitochondria of the cells has a fluorescence background. This is due to the relatively small Stokes shift and the wide half-peak width. This results in the measured signal being inaccurate. Specifically, the existing technology has the following defects:

[0004] The first approach is to chemically synthesize gold nanorods in vitro and then modify their surfaces to capture tumor cells. This method is extremely complex, has poor controllability, and is inefficient in capturing tumors. The surface-enhanced Raman spectroscopy signal is unstable, and it cannot produce near-infrared fluorescence at adjustable concentrations for tumor visualization.

[0005] Secondly, although sodium citrate can be used to synthesize gold nanoparticles in vitro in the existing technology, it is relatively difficult for circulating tumor cells to specifically bind to gold nanoparticles.

[0006] Third, surface-enhanced Raman spectroscopy can only be performed on body fluids and cannot be used to detect solid tumors.

[0007] Fourth, the gold-silver nanocore-shell structure synthesized in vitro has complex synthesis steps, and the silver nanostructure is easily oxidized, affecting the stability of Raman spectroscopy. In addition, mi-RNA is usually present in the cytoplasm of tumor cells, so it is difficult for nanoprobes to detect substances in tumor cells. Only when the cells are alive and the cells actively endocytose into the cells, can tumor cells be detected. However, this process is extremely long, and there may be the presence of protein coronas or other substances that cover the specific targets modified on the surface of the nanomaterials, making it difficult to measure specific tumor cell surface enhanced Raman spectroscopy signals.

[0008] Fifth, graphene is used to reduce gold nanoparticles inside cells. It should be noted that graphene itself has reducing properties, and it is through graphene that gold is reduced. Moreover, since graphene has surface-enhanced Raman spectroscopy characteristics, it is impossible to determine the specific structure that is enhanced.

[0009] Sixth, while surface-enhanced Raman spectroscopy (SERS) measurements of gold nanoparticles generated in 3T3 fibroblasts cannot be performed on tumor cells, as gold nanoparticles are not generated in tumor cells. The resulting Raman spectroscopy signals are not uniform and stable.

[0010] Seventh, gold nanoparticles are produced in the cell nucleus, but there are no surface-enhanced Raman spectroscopy features in the cell nucleus that distinguish tumor cells from normal cells. Furthermore, it is not possible to perform Raman spectroscopy analysis on tissues, and to achieve near-infrared fluorescence imaging of tumor cells / tissues through adjustable concentrations.

[0011] Eighth, the gold nanoparticles synthesized from the extract of lemongrass plants are not synthesized inside cells, and the synthesized gold nanostructure is a kind of gold triangle sheet with the (111) crystal plane of gold on the surface, and the surface enhanced Raman spectroscopy effect is relatively poor.

[0012] Finally, the sides and slopes of the triangular nanoplates of the nanobelts exhibit plasmon excitons in the quadrupole resonance mode in the infrared region, but the synthesis time of this scheme is long. The gold nanostructure is a kind of gold triangular sheet with the (111) crystal plane of gold on the surface. The surface-enhanced Raman spectroscopy effect is relatively poor, with only a strong photothermal effect. It cannot effectively generate surface-enhanced Raman signals and cannot realize the determination and detection of tumor cells. Summary of the Invention

[0013] Technical Problems Solved: In response to the above-mentioned problems, the present invention provides a combination reagent for multidimensional tumor detection based on in situ intracellular mineralization and its application. By adjusting the concentration and incubation time of the combination reagent, near-infrared emitting gold nanostructures with ultra-narrow half-width and ultra-long Stokes shift, or plasmon resonance gold nanostructures (nanogold with surface-enhanced Raman spectroscopy properties) are generated in tumor cells. Those skilled in the art can identify tumors and non-tumors through this fluorescence phenomenon. In addition, the plasmon resonance gold nanostructures generated in tumor cells can be used to further measure their Raman spectra, and tumor cells and non-tumor cells (or tumor tissues and non-tumor tissues) can be identified by PCA analysis.

[0014] Technical solution: A multidimensional tumor detection combination reagent based on in situ intracellular mineralization includes reagent 1 and reagent 2, wherein reagent 1 is chloroauric acid and reagent 2 is ultrapure water containing sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate.

[0015] The concentration of the above-mentioned chloroauric acid is 20 μM-1000 μM.

[0016] Preferably, the concentration of the chloroauric acid is 50 μM-100 μM.

[0017] Also included are stimulating drugs, which are hydrochloric acid, heparin, polyethylene glycol or triglyceride.

[0018] In the above reagent 2, 6-9 g of sodium chloride, 0.2 g-0.5 g of potassium chloride, 1 g-3 g of disodium hydrogen phosphate, and 0 g-2 g of potassium dihydrogen phosphate are mixed with every 100 mL of ultrapure water.

[0019] Preferably, in the above reagent 2, 6 g of sodium chloride, 0.3 g of potassium chloride, 1 g of disodium hydrogen phosphate, and 1 g of potassium dihydrogen phosphate are mixed with every 100 mL of ultrapure water.

[0020] The mass ratio of the above reagent 1 and reagent 2 is 1:(200-2000).

[0021] Preferably, the mass ratio of the above reagent 1 and reagent 2 is 1:1000.

[0022] The application of the above-mentioned combination reagents in the preparation of multidimensional tumor detection products based on in situ intracellular mineralization.

[0023] A multidimensional tumor detection product based on in situ intracellular mineralization, the active ingredient of which is the above-mentioned combination reagent.

[0024] Beneficial effects: First, the technical solution of the present invention is different from the existing technology in which gold nanostructures are synthesized in vitro and then tumor cells are captured and identified by other specific compound molecules. The present invention synthesizes gold nanostructures in tumor cells through liquid-liquid phase separation, has good specificity, and does not require an in vitro synthesis step, which is convenient and simple. Secondly, the synthesis of the present invention is convenient and fast, with very low cost, and does not require the addition of other hazardous chemicals. Third, the simple operation of the present invention can achieve the labeling of tumor tissue / cell near-infrared fluorescence and molecular recognition and detection by surface-enhanced Raman spectroscopy. Finally, the present invention has high repeatability and can achieve analysis, identification and imaging of chemical molecules of tumor cells at the single-cell level. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a transmission electron microscopy image of gold nanoparticles;

[0026] Figure 2 is the fluorescence spectrum of gold nanostructure;

[0027] Figure 3 This is the surface-enhanced Raman spectrum of tumor tissue;

[0028] Figure 4 PCA classification of tumor tissue and normal tissue. DETAILED DESCRIPTION

[0029] The present invention will be described in further detail below with reference to the accompanying drawings and specific implementation methods.

[0030] Example 1:

[0031] 1. Prepare solution 1 (chloroauric acid solution) and solution 2 (sodium chloride, potassium chloride, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and liquid-liquid phase separation stimulating drug) of different concentrations. The specific ingredients are as follows:

[0032] Solution 1

[0033] name dose chloroauric acid 20μm-1000μm

[0034] Solution 2

[0035] name dose Sodium chloride 6g-9g potassium chloride 0.2g-0.5g Disodium hydrogen phosphate 1g-3g Potassium dihydrogen phosphate 0g-2g Ultrapure water 100mL Overall system 100mL

[0036] 2. Take solution 1 and solution 2 separately, mix them, add them to cells or tumor tissue, soak for 6 hours to 5 days, and then analyze the fluorescence phenomenon and Raman spectral characteristics of the tumor tissue / cells using a fluorescence microscope, a stereo fluorescence microscope, and a Raman spectrometer.

[0037] 3. The Raman spectra of tumor tissue / cells and normal tissue / cells are classified and modeled using PCA, and then tumor cells / tissues are detected.

[0038] Principles of intracellular synthesis of fluorescent gold nanoprobes and surface-enhanced Raman effect gold nanostructures:

[0039] After adding Solution 1 and Solution 2 to tumor cells, liquid-liquid phase separation occurs. Gold ions nucleate at the β-turns of the protein secondary structure in the droplets produced by this phase separation, forming gold clusters. These clusters then grow through protein adsorption on different crystal surfaces, ultimately forming gold nanostructures with near-infrared luminescence and surface-enhanced Raman effects. This method, combined with fluorescence imaging and Raman spectral signals from tumor cells and tissues, allows for tumor detection and analysis through PCA classification. Liquid-liquid phase separation does not occur in normal cells / tissues, and therefore gold nanostructures cannot be generated in normal cells.

[0040] result:

[0041] 1. Transmission electron microscopy images of gold nanostructures ( Figure 1 ), showing relatively uniform gold nanoparticles with a diameter of approximately 30 nm. Gold nanostructures smaller than 30 nm exhibit fluorescence, while those larger than 30 nm exhibit surface-enhanced Raman effects.

[0042] 2. Fluorescence spectrum of gold nanostructures ( Figure 2 ), it was found that the optimal excitation wavelength of the gold nanostructure was 570nm and the optimal emission wavelength was 855, the half-peak width of the emission wavelength was 17nm, and the Stokes shift was 285nm.

[0043] 3. Surface enhanced Raman spectroscopy of tumor tissue ( Figure 3 ) Tumor tissue has good surface enhanced Raman characteristics, which can well display the chemical molecular characteristics of tumor tissue.

[0044] 4. PCA classification of tumor tissue and normal tissue ( Figure 4 ), with an accuracy rate of greater than 98% for tumor tissue detection.

Claims

1. A multidimensional tumor detection combination reagent based on in situ intracellular mineralization, characterized in that: The method comprises reagent 1 and reagent 2, wherein reagent 1 is chloroauric acid and reagent 2 is ultrapure water containing sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate.

2. The multidimensional tumor detection combination reagent based on in situ intracellular mineralization according to claim 1, characterized in that: The concentration of the chloroauric acid is 20 μM-1000 μM.

3. The multidimensional tumor detection combination reagent based on in situ intracellular mineralization according to claim 2, characterized in that: The concentration of the chloroauric acid is 50 μM-100 μM.

4. The multidimensional tumor detection combination reagent based on in situ intracellular mineralization according to claim 1, characterized in that: Also included are stimulating drugs, which are hydrochloric acid, heparin, polyethylene glycol or triglyceride.

5. The multidimensional tumor detection combination reagent based on in situ intracellular mineralization according to claim 1, characterized in that: In the reagent 2, 6-9 g of sodium chloride, 0.2 g-0.5 g of potassium chloride, 1 g-3 g of disodium hydrogen phosphate, and 0 g-2 g of potassium dihydrogen phosphate are mixed per 100 mL of ultrapure water.

6. The multidimensional tumor detection combination reagent based on in situ intracellular mineralization according to claim 5, characterized in that: In the reagent 2, 6 g of sodium chloride, 0.3 g of potassium chloride, 1 g of disodium hydrogen phosphate, and 1 g of potassium dihydrogen phosphate were mixed per 100 mL of ultrapure water.

7. The multidimensional tumor detection combination reagent based on in situ intracellular mineralization according to claim 1, characterized in that: The mass ratio of the reagent 1 to the reagent 2 is 1:(200-2000).

8. The multidimensional tumor detection combination reagent based on in situ intracellular mineralization according to claim 7, characterized in that: The mass ratio of the reagent 1 to the reagent 2 is 1:1000.

9. Use of the combination reagent according to any one of claims 1 to 8 in the preparation of a multidimensional tumor detection product based on in situ intracellular mineralization.

10. A multidimensional tumor detection product based on in situ intracellular mineralization, characterized by: The active ingredient is the combined agent according to any one of claims 1 to 8.

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