Preparation method and application of a magnetic nanosensor for early diagnosis of tumors

Abstract

The present invention prepares a magnetic nanosensor that can be used for early diagnosis of tumors, specifically a magnetic nanosensor FSC-D-P0 that can be used for circulating tumor cell analysis and tumor nuclear magnetic resonance / fluorescence dual-modal imaging. Among them, FSC-D-P0 is obtained by modifying the Fe3O4 core and the silicon-carbon doped nanolayer SiO2 / C shell through double-stranded DNA-mediated Cy5-labeled aptamer D-P0. FSC-D-P0 can bind to the MUC1 protein on the surface of the tumor to complete the capture of tumor cells. Under the interaction between the silicon-carbon doped nanolayer SiO2 / C and the cells, fluorescence is restored to achieve visualization of circulating tumor cells. Then, under the action of an external magnetic field, tumor cells are enriched, and under the action of the endonuclease EcoR1, the captured circulating tumor cells are released. At the same time, based on the active targeting of magnetic nanoparticles and aptamers, specific fluorescence recovery and MRI imaging, FSC-D-P0 can avoid false positives caused by non-targeted accumulation of magnetic nanoparticles in tissues, thereby accurately completing tumor imaging.

Description

Technical Field

[0001] The present invention belongs to the field of materials chemistry and relates to a magnetic nanosensor that can be used for early diagnosis of tumors, and specifically to a preparation method and application of a magnetic nanosensor that can be used for circulating tumor cell analysis and tumor nuclear magnetic resonance / fluorescence dual-modality imaging. Background Art

[0002] Diagnosing and treating cancer at its early stages is a key approach to cancer prevention. Currently, cancer diagnosis is primarily performed clinically through imaging techniques combined with tissue biopsies. However, mainstream imaging technologies, such as magnetic resonance imaging (MRI), typically have low sensitivity and are unable to identify small benign or malignant tumor lesions, making them of limited value for early cancer diagnosis. Furthermore, the non-targeted accumulation of contrast agents often results in false-positive imaging results, hindering clinicians' ability to accurately diagnose tumors. Therefore, improving the targeting and sensitivity of existing imaging technologies for imaging tumor tissue is a major challenge facing the improvement of clinical tumor diagnosis.

[0003] In recent years, biosensors based on the fluorescence quenching-recovery mechanism have shown great potential in cancer detection research due to their ease of synthesis and specific targeting. However, their sensitivity is often affected by the mismatched interactions between the quencher and the fluorophore. In addition, due to instability and non-targeted delivery, these biosensors rarely achieve accurate in vivo fluorescence imaging. At the same time, tissue biopsy, as a recognized and accurate method for cancer detection, is hindered from widespread clinical application due to its invasiveness and poor patient tolerance. Therefore, increasing the targeted delivery of contrast agents to avoid their nonspecific accumulation and regulating biosensing interactions to improve their imaging sensitivity remain difficult problems facing cancer detection and imaging research.

[0004] The emerging technology of liquid biopsy has attracted attention due to its non-invasive nature and significant clinical significance. However, circulating tumor cells in the complex blood environment are scarce and difficult to capture, purify, and detect using detection instruments, which greatly hinders the progress of liquid biopsy research. Moreover, tumor imaging and biopsy are currently performed using different platforms and probes, which significantly increases the complexity and cost of detection and affects the consistency of results. Therefore, how to integrate the capture, detection, and release of circulating tumor cells and the precise imaging of tumors into a single monitoring system is a problem that researchers need to solve.

[0005] Therefore, in the present invention, a novel magnetic nanosensor, FSC-D-P0, is constructed based on magnetic nanoparticles by silicon-carbon doping and ligand modification of their surfaces. This sensor can bind to the MUC1 protein on the tumor surface, capture tumor cells, and restore fluorescence under the interaction between the silicon-carbon doped nanolayer SiO2 / C and the cells, enabling visualization of circulating tumor cells. Subsequently, under the action of an external magnetic field, tumor cells are enriched, and under the action of the endonuclease EcoR1, the captured circulating tumor cells are released. Simultaneously, based on the active targeting of the magnetic nanoparticles and aptamers, specific fluorescence recovery, and MRI imaging, FSC-D-P0 can avoid false positives caused by the non-targeted accumulation of magnetic nanoparticles in tissues. FSC-D-P0 combines the capture, detection, and release of circulating tumor cells with dual-modality tumor nuclear magnetic resonance / fluorescence imaging on a single platform, enabling multi-faceted tumor diagnosis in vitro and in vivo, and showing great potential for application in clinical tumor diagnosis and treatment. Summary of the Invention

[0006] The present invention aims to address the shortcomings of existing clinical tumor detection methods by providing a magnetic nanosensor for early tumor diagnosis. Specifically, it relates to a method for preparing and applying a magnetic nanosensor that can be used for circulating tumor cell analysis and dual-modality nuclear magnetic resonance / fluorescence imaging of tumors, comprising the following reaction steps:

[0007] Step (1): using a trivalent iron compound such as FeCl3·6H2O as a raw material and diethylene glycol and ethylene glycol as a reaction medium to prepare magnetic nanoparticles Fe3O4;

[0008] Step (2): using resorcinol, formaldehyde and tetrapropoxymethane as raw materials to carry out surface silicification of magnetic nanoparticles Fe3O4 to prepare silicified magnetic nanoparticles Fe3O4@SiO2 / P, wherein the mass ratio of Fe3O4 to resorcinol is 100:1-30:1, the mass ratio of Fe3O4 to formaldehyde is 100:1-40:1, and the mass ratio of Fe3O4 to tetrapropoxymethane is 100:1-20:1;

[0009] Step (3): Carbonizing the Fe3O4@SiO2 / P in step (2) by high temperature and high pressure reaction under nitrogen atmosphere to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C, the reaction temperature is above 500°C, and the reaction time is not less than 4 hours;

[0010] Step (4): oxidizing the surface of the carbonized magnetic nanoparticles Fe3O4@SiO2 / C by an oxidant to obtain surface-modifiable magnetic nanoparticles FSC. The oxidant may include strong oxidants such as hydrogen peroxide and potassium permanganate.

[0011] Step (5): Based on the FSC in step (4), the magnetic nanosensor FSC-D-P0 was prepared by surface modification of ssDNA1 and ssDNA2-P0, and the ratio of aptamer D-P0 to FSC (nmol / mg) was 0.1:1 to 0.8:1;

[0012] The magnetic nanosensor FSC-D-P0 provided by the present invention can bind to the MUC1 protein on the surface of the tumor to complete the capture of tumor cells. Under the interaction between the silicon-carbon doped nanolayer SiO2 / C and the cells, the fluorescence on the surface of the tumor cells is restored, realizing the visualization of circulating tumor cells. Then, under the mediation of an external magnetic field, the enrichment of fluorescently labeled tumor cells is completed. At the same time, without affecting the viability of the cells, the captured circulating tumor cells are released through the action of the endonuclease EcoR1. At the same time, based on the active targeting of magnetic nanoparticles and aptamers, specific fluorescence recovery and nuclear magnetic fluorescence dual-modal imaging, FSC-D-P0 can avoid MRI false positives caused by the non-targeted accumulation of magnetic nanoparticles in tissues, thereby achieving accurate tumor imaging.

[0013] The magnetic nanosensor FSC-D-P0 provided by the present invention has a Fe3O4 precursor which is a compound that can provide trivalent iron.

[0014] The magnetic nanosensor FSC-D-P0 provided by the present invention uses reagents for oxidizing the silicon-carbon-doped nanolayer SiO2 / C, including but not limited to strong oxidants such as hydrogen peroxide and potassium permanganate.

[0015] The magnetic nanosensor FSC-D-P0 provided by the present invention has a reaction temperature for carbonizing the silicon-carbon doped nanolayer SiO2 / C at a temperature above 500°C and a reaction time of not less than 4 hours.

[0016] In the magnetic nanosensor FSC-D-P0 provided by the present invention, the fluorescence quenching and recovery of Cy5 are mainly controlled by the silicon-carbon doped nanolayer SiO2 / C.

[0017] The magnetic nanosensor FSC-D-P0 provided by the present invention can capture and enrich circulating tumor cells and release them in response to the endonuclease EcoR1, and this process does not affect the vitality of circulating tumor cells.

[0018] The magnetic nanosensor FSC-D-P0 provided by the present invention can achieve accurate imaging of tumor tissue through the dual-modality imaging mode of nuclear magnetic resonance (MRI) and fluorescence fluorescence (FL), thus avoiding the occurrence of false positives in diagnosis.

[0019] The magnetic nanosensor FSC-D-P0 provided by the present invention is applicable to tumors expressing MUC1 protein, including but not limited to pancreatic cancer, breast cancer, ophthalmic tumors, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : TEM image of FSC (Bar=100 nm).

[0021] Figure 2 : STEM image of FSC-D-P0.

[0022] Figure 3 : Infrared spectroscopic analysis of FSC-D-P0 and intermediates (Fe3O4, Fe3O4@SiO2 / C and FSC).

[0023] Figure 4 :T2 relaxation rate of FSC and FSC-D-P0 (A) and T2-MRI images (B) and FSC-D-P0 dispersion images before and after magnetic interference (C)

[0024] Figure 5 : Fluorescence spectra and time-dependent cellular uptake behavior of FSC@D-P0, FSC(-Si)@D-P0, and FSC(-C)@D-P0. Inset: Aqueous solution images of FSC, FSC(-Si), and FSC(-C).

[0025] Figure 6 : Time-dependent fluorescence recovery images of FSC@D-P0 in MCF-7 and MCF-10A.

[0026] Figure 7 : Time-dependent fluorescence recovery images of FSC@D-P0 in PANC-1 and HPDE6C7.

[0027] Figure 8 Fluorescence spectra of FSC-D-P0 cells co-incubated with different concentrations of MCF-7 cells in (A) PBS, (B) PBS containing 4% serum, and (C) blood, and (D) fluorescence stability evaluation. Inset: Linear regression equation between cell number and fluorescence intensity.

[0028] Figure 9 : Cell capture efficiency of FSC-D-P0 at different incubation times.

[0029] Figure 10 :Image of FSC-D-P0 interaction with cells and Live / Dead viability evaluation of released cells.

[0030] Figure 11 : Fluorescence spectra of FSC-D-P0 after incubation with blood from tumor-bearing and normal mice. Inset: FSC-D-P0, calculated by the linear regression equation between the cell number in blood and the fluorescence intensity, can be used to detect the number of circulating tumor cells.

[0031] Figure 12: T2-MRI images (6 h after administration) and in vivo and ex vivo fluorescence images (10 h after administration) of tumor-bearing mice under different intervention conditions.

[0032] Figure 13 : CCK-8 cell viability evaluation of MCF-7 and MCF-10A cells after incubation with different concentrations of FSC-D-P0 for 2 h. DETAILED DESCRIPTION

[0033] Example 1

[0034] 0.6 g FeCl3·6H2O, 1.5 g NaAc, 1.5 g sodium acrylate were mixed with 5 mL diethylene glycol and 15 mL ethylene glycol, heated and stirred for 30 min, and then transferred to an autoclave for reaction (200°C / 10 h). After alternate washing with ethanol and water, magnetic nanoparticles Fe3O4 were obtained.

[0035] Example 2

[0036] 0.8 g FeCl3·6H2O, 1.5 g NaAc, 1.5 g sodium acrylate were mixed with 5 mL diethylene glycol and 15 mL ethylene glycol, heated and stirred for 30 min, and then transferred to an autoclave for reaction (200°C / 10 h). After alternate washing with ethanol and water, magnetic nanoparticles Fe3O4 were obtained.

[0037] Example 3

[0038] 1 g FeCl3·6H2O, 1.5 g NaAc, 1.5 g sodium acrylate were mixed with 5 mL diethylene glycol and 15 mL ethylene glycol, heated and stirred for 30 min, and then transferred to an autoclave for reaction (200°C / 8 h). After alternate washing with ethanol and water, magnetic nanoparticles Fe3O4 were obtained.

[0039] Example 4

[0040] 1.2 g FeCl3·6H2O, 1.5 g NaAc, 1.5 g sodium acrylate were mixed with 5 mL diethylene glycol and 15 mL ethylene glycol, heated and stirred for 30 min, and then transferred to an autoclave for reaction (200°C / 8 h). After alternate washing with ethanol and water, magnetic nanoparticles Fe3O4 were obtained.

[0041] Example 5

[0042] 1.4 g FeCl3·6H2O, 1.5 g NaAc, 1.5 g sodium acrylate were mixed with 5 mL diethylene glycol and 15 mL ethylene glycol, heated and stirred for 30 min, and then transferred to an autoclave for reaction (200°C / 8 h). After alternate washing with ethanol and water, magnetic nanoparticles Fe3O4 were obtained.

[0043] Example 6

[0044] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1 mg of resorcinol was mixed with 1 mg of formaldehyde and 1 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0045] Example 7

[0046] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1.5 mg of resorcinol was mixed with 1 mg of formaldehyde and 1 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0047] Example 8

[0048] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2 mg of resorcinol was mixed with 1 mg of formaldehyde and 1 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0049] Example 9

[0050] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2.5 mg of resorcinol was mixed with 1 mg of formaldehyde and 1 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0051] Example 10

[0052] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1 mg of resorcinol was mixed with 1.5 mg of formaldehyde and 1 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0053] Example 11

[0054] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1.5 mg of resorcinol was mixed with 1.5 mg of formaldehyde and 1 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0055] Example 12

[0056] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2 mg of resorcinol was mixed with 1.5 mg of formaldehyde and 1 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0057] Example 13

[0058] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2.5 mg of resorcinol was mixed with 1.5 mg of formaldehyde and 1 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0059] Example 14

[0060] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1 mg of resorcinol was mixed with 2 mg of formaldehyde and 1 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0061] Example 15

[0062] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1.5 mg of resorcinol was mixed with 2 mg of formaldehyde and 1 mg of tetrapropoxymethane and reacted for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0063] Example 16

[0064] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2 mg of resorcinol was mixed with 2 mg of formaldehyde and 1 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0065] Example 17

[0066] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2.5 mg of resorcinol was mixed with 2 mg of formaldehyde and 1 mg of tetrapropoxymethane and reacted for 24 hours to obtain siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0067] Example 18

[0068] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1 mg of resorcinol was mixed with 1 mg of formaldehyde and 1.5 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0069] Example 19

[0070] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1.5 mg of resorcinol was mixed with 1 mg of formaldehyde and 1.5 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0071] Example 20

[0072] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2 mg of resorcinol was mixed with 1 mg of formaldehyde and 1.5 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0073] Example 21

[0074] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2.5 mg of resorcinol was mixed with 1 mg of formaldehyde and 1.5 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0075] Example 22

[0076] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1 mg of resorcinol was mixed with 1.5 mg of formaldehyde and 2 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0077] Example 23

[0078] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1.5 mg of resorcinol was mixed with 1.5 mg of formaldehyde and 2 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0079] Example 24

[0080] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2 mg of resorcinol was mixed with 1.5 mg of formaldehyde and 2 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0081] Example 25

[0082] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2.5 mg of resorcinol was mixed with 1.5 mg of formaldehyde and 2 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0083] Example 26

[0084] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1 mg of resorcinol was mixed with 2 mg of formaldehyde and 2.5 mg of tetrapropoxymethane and reacted for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0085] Example 27

[0086] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 1.5 mg of resorcinol was mixed with 2 mg of formaldehyde and 2.5 mg of tetrapropoxymethane and reacted for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0087] Example 28

[0088] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2 mg of resorcinol was mixed with 2 mg of formaldehyde and 2.5 mg of tetrapropoxymethane and reacted for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. Furthermore, the Fe3O4@SiO2 / C was oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0089] Example 29

[0090] 80 mg of Fe3O4 prepared in Example 1 was added to 70 mL of ethanol, 10 mL of water, and 3 mL of ammonia. After complete ultrasonic dispersion, 2.5 mg of resorcinol was mixed with 2 mg of formaldehyde and 2.5 mg of tetrapropoxymethane for 24 hours to produce siliconized magnetic nanoparticles Fe3O4@SiO2 / P. The Fe3O4@SiO2 / P was then calcined at 700°C under a nitrogen atmosphere for 5 hours to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C. The Fe3O4@SiO2 / C was further oxidized with 10% hydrogen peroxide to obtain surface-modified magnetic nanoparticles FSC.

[0091] Example 30

[0092] Equal moles of ssDNA1 and ssDNA2-P0 were mixed with STE buffer, heated in an oil bath at 95°C for 5 minutes, and allowed to cool freely to room temperature to form double-stranded DNA (dsDNA, D-P0). Simultaneously, the FSC prepared in Example 6 was resuspended in 2.5 mg / mL ultrapure water and the pH was adjusted to 6.0. After the addition of EDC and NHS, the medium was activated by vortex mixing at 25°C for 30 minutes. Then, D-P0 was added (D-P0:FSC nmol / mg = 0.14), and the reaction was continued overnight at 25°C. The product, FSC-D-P0, was purified by multiple magnetic enrichment.

[0093] Example 31

[0094] The same molar amount of ssDNA1 and ssDNA2-P0 was mixed with STE buffer, heated in an oil bath at 95°C for 5 minutes, and allowed to cool freely to room temperature to form double-stranded DNA (dsDNA, D-P0). Simultaneously, the FSC prepared in Example 6 was resuspended in 2.5 mg / mL ultrapure water and the pH was adjusted to 6.0. After the addition of EDC and NHS, the medium was activated by vortex mixing at 25°C for 30 minutes. Then, D-P0 was added (D-P0:FSC nmol / mg = 0.28), and the reaction was continued overnight at 25°C. The product FSC-D-P0 was purified by multiple magnetic enrichment.

[0095] Example 32

[0096] The same molar amount of ssDNA1 and ssDNA2-P0 was mixed with STE buffer, heated in an oil bath at 95°C for 5 minutes, and allowed to cool freely to room temperature to form double-stranded DNA (dsDNA, D-P0). Simultaneously, the FSC prepared in Example 6 was resuspended in 2.5 mg / mL ultrapure water and the pH was adjusted to 6.0. After the addition of EDC and NHS, the medium was activated by vortex mixing at 25°C for 30 minutes. Then, D-P0 was added (D-P0:FSC nmol / mg = 0.56), and the reaction was continued overnight at 25°C. The product FSC-D-P0 was purified by multiple magnetic enrichment.

[0097] Example 33

[0098] Equal moles of ssDNA1 and ssDNA2-P0 were mixed with STE buffer, heated in an oil bath at 95°C for 5 minutes, and allowed to cool freely to room temperature to form double-stranded DNA (dsDNA, D-P0). Simultaneously, the FSC prepared in Example 6 was resuspended in 2.5 mg / mL ultrapure water and the pH was adjusted to 6.0. After the addition of EDC and NHS, the medium was activated by vortex mixing at 25°C for 30 minutes. Then, D-P0 was added (D-P0:FSC nmol / mg = 0.84), and the reaction was continued overnight at 25°C. The product, FSC-D-P0, was purified by multiple magnetic enrichment.

[0099] Example 34

[0100] The same mass of ssDNA1 and ssDNA2-P0 was mixed with STE buffer, heated in an oil bath at 95°C for 5 minutes, and allowed to cool freely to room temperature to form double-stranded DNA (dsDNA, D-P0). At the same time, the FSC prepared in Example 6 was resuspended in 2.5 mg / mL ultrapure water and the pH was adjusted to 6.0. After the addition of EDC and NHS, the medium was activated by vortex mixing at 25°C for 30 minutes. Then, D-P0 was added (D-P0:FSCnmol / mg=0.14), and the reaction was continued overnight at 25°C. The product FSC-D-P0 was purified by multiple magnetic enrichment.

[0101] Example 35

[0102] The same mass of ssDNA1 and ssDNA2-P0 was mixed with STE buffer, heated in an oil bath at 95°C for 5 minutes, and allowed to cool freely to room temperature to form double-stranded DNA (dsDNA, D-P0). At the same time, the FSC prepared in Example 6 was resuspended in 2.5 mg / mL ultrapure water and the pH was adjusted to 6.0. After the addition of EDC and NHS, the medium was activated by vortex mixing at 25°C for 30 minutes. Then, D-P0 was added (D-P0:FSCnmol / mg=0.28), and the reaction was continued overnight at 25°C. The product FSC-D-P0 was purified by multiple magnetic enrichment.

[0103] Example 36

[0104] The same mass of ssDNA1 and ssDNA2-P0 was mixed with STE buffer, heated in an oil bath at 95°C for 5 minutes, and allowed to cool freely to room temperature to form double-stranded DNA (dsDNA, D-P0). At the same time, the FSC prepared in Example 6 was resuspended in 2.5 mg / mL ultrapure water and the pH was adjusted to 6.0. After the addition of EDC and NHS, the medium was activated by vortex mixing at 25°C for 30 minutes. Then, D-P0 was added (D-P0:FSCnmol / mg=0.56), and the reaction was continued overnight at 25°C. The product FSC-D-P0 was purified by multiple magnetic enrichment.

[0105] Example 37

[0106] The same mass of ssDNA1 and ssDNA2-P0 was mixed with STE buffer, heated in an oil bath at 95°C for 5 minutes, and allowed to cool freely to room temperature to form double-stranded DNA (dsDNA, D-P0). At the same time, the FSC prepared in Example 6 was resuspended in 2.5 mg / mL ultrapure water and the pH was adjusted to 6.0. After the addition of EDC and NHS, the medium was activated by vortex mixing at 25°C for 30 minutes. Then, D-P0 was added (D-P0:FSCnmol / mg=0.84), and the reaction was continued overnight at 25°C. The product FSC-D-P0 was purified by multiple magnetic enrichment.

[0107] Example 38

[0108] The FSC-D-P0 prepared in Example 30 was observed by TEM. The results showed that the nanoparticles were composed of materials with different properties, had a core-shell structure, and were uniform in size with a particle size of about 200 nm. Figure 1 .

[0109] Example 39

[0110] STEM observation of the FSC-D-PO prepared in Example 30 showed that the nanoparticles were composed of iron, silicon, oxygen, carbon, and phosphorus. Among them, the iron element was located in the core layer, and the other elements were distributed in the shell layer, indicating that the nanoparticles were composed of a Fe3O4 core and a silicon-carbon doped shell structure, and that D-PO was successfully modified. Figure 2 .

[0111] Example 40

[0112] The bonding forms of Fe3O4 prepared in Example 1, FSC and Fe3O4@SiO2 / C prepared in Example 6, and FSC-D-PO prepared in Example 30 were compared by FTIR. The results showed that the oxidation of hydrogen peroxide provided modifiable groups (-COOH and -OH) on the surface of FSC. Then, through the reaction of -COOH and -NH2 (v N-H 3135cm -1 ,δ N-H 1650-1250cm -1 and v C-N 1310-1200cm -1 The Cy5-labeled double-stranded DNA modified P0 aptamer (D-P0) was successfully modified on the FSC surface to obtain FSC-D-P0. Figure 3 .

[0113] Example 41

[0114] The T2 imaging properties of FSC-D-P0 prepared in Example 30 were investigated by nuclear magnetic resonance scanning. The results showed that the water dispersibility and stability of FSC-D-P0 were significantly improved due to the modification of D-P0, and the FSC-D-P0 had a higher longitudinal relaxivity (r1 = 104.18 mM -1 S -1 ) and thus mediate high-resolution T2 nuclear magnetic imaging. At the same time, FSC-D-P0 has strong magnetic targeting properties. Under the induction of an external magnetic field, it can complete enrichment in a short time. Please see the attached Figure 4 .

[0115] Example 42

[0116] The fluorescence recovery characteristics of silicon-carbon doped FSC and non-covalently linked nanocarriers FSC(-Si) and FSC(-C) were compared by fluorescence spectrometry and cellular level. The results showed that the pure carbon structure FSC(-Si)@D-P0 completely adsorbed D-P0 and completely quenched the Cy5 fluorescence, while the pure silicon structure FSC(-C)@D-P0 had a weak interaction with D-P0 and almost no quenching of Cy5. In FSC@D-P0, the composite SiO2 doped C structure enables FSC to have a better interaction with the fluorescent aptamer, which can properly control the fluorescence quenching and recovery of Cy5, and then illuminate tumor cells with the extension of incubation time. Please see the attached figure. Figure 5 .

[0117] Example 43

[0118] The fluorescence imaging properties of FSC-D-P0 were investigated using pancreatic cancer cells and breast cancer cells as models. The results showed that due to the aptamer-mediated tumor cell targeting and fluorescence quenching recovery biosensing properties, the Cy5 fluorescence of FSC-D-P0 gradually recovered in MCF-7 tumor cells with increasing co-incubation time, while the fluorescence of normal cells continued to be quenched. Furthermore, this fluorescence recovery property is applicable to all tumor cells that overexpress MUC1. (See attached figure for details.) Figure 6 and attached Figure 7 .

[0119] Example 44

[0120] Through fluorescence spectral analysis, using breast cancer cells as a model, the capture and fluorescence recovery characteristics of circulating tumor cells by FSC-D-P0 were investigated. The results showed that FSC-D-P0 could capture tumor cells and complete fluorescence recovery in different media. Moreover, even in media with very few cells, there was a good linear correlation between the number of captured tumor cells and the fluorescence value. At the same time, the fluorescence recovery stability of the cell surface was good, which could achieve accurate and stable capture of tumor cells. Figure 8 .

[0121] Example 44

[0122] By fluorescence spectroscopy analysis, the efficiency of FSC-D-P0 in capturing tumor cells was investigated using breast cancer cells as a model. The results showed that after 30 minutes of co-incubation, FSC-D-P0 was able to capture more than 90% of tumor cells. Figure 9 .

[0123] Example 45

[0124] The cellular process of FSC-D-P0 fluorescence capture was studied using TEM and Live / Dead-mediated cell viability probes. The results showed that under the mediation of the P0 aptamer, FSC-D-P0 could specifically adsorb to the surface of tumor cells, and then enrich tumor cells under the action of a magnetic field. Then, under the action of EcoR1 primers, the captured tumor cells were released without affecting cell viability. See the attached figure. Figure 10 .

[0125] Example 46

[0126] Through fluorescence spectroscopy analysis, the FSC-D-P0 assay was used to investigate the specific capture rate of circulating tumor cells using mice bearing breast cancer at different growth stages. The results showed that FSC-D-P0 has high specificity and strong anti-interference ability in detecting CTCs. It also has a relatively sensitive response to CTCs in the blood of MCF-7 tumor-bearing mice, and the fluorescence is positively correlated with the CTC concentration and the number of days of tumor growth. (See attached figure for details.) Figure 11 .

[0127] Example 47

[0128] The dual-modal imaging properties of FSC-D-P0 were investigated using tumor-bearing mice as a model using in vivo small animal imaging and magnetic resonance imaging. The results showed that due to the tumor targeting mediated by the P0 aptamer and the magnetic targeting properties mediated by Fe3O4, FSC-D-P0 achieved significant T2 imaging of the tumor 6 hours after injection. Furthermore, 10 hours after injection, FSC-D-P0 also mediated Cy5 fluorescence imaging of the tumor tissue. The combination of MRI and fluorescence imaging avoids false positives due to magnetic nanoparticle accumulation and improves the specificity of tumor imaging. (See attached figure for details.) Figure 12 .

[0129] Example 48

[0130] The biosafety of FSC-D-P0 was investigated using CCK-8. The results showed that after 2 hours of co-incubation with tumor cells or normal cells, more than 90% of the cells survived, indicating that FSC-D-P0 has low toxicity and can be used as a nanocarrier for the detection and imaging of tumor cells. Please see the attached Figure 13 .

Claims

1. A magnetic nanosensor FSC-D-P0 that can be used for circulating tumor cell analysis and diagnosis and tumor nuclear magnetic resonance / fluorescence dual-modality imaging, characterized in that: The preparation method of the magnetic nanosensor FSC-D-P0 is as follows: Step (1): using FeCl3·6H2O as raw material and diethylene glycol and ethylene glycol as reaction medium to prepare magnetic nanoparticles Fe3O4; Step (2): Silica-treated magnetic nanoparticles Fe3O4 were prepared by using resorcinol, formaldehyde and tetrapropoxymethane as raw materials to prepare silica-treated magnetic nanoparticles Fe3O4@SiO2 / P, wherein the mass ratio of Fe3O4 to resorcinol was 80:1, the mass ratio of Fe3O4 to formaldehyde was 80:1, and the mass ratio of Fe3O4 to tetrapropoxymethane was 80:

1. Step (3): Carbonizing the Fe3O4@SiO2 / P in step (2) by high temperature and high pressure reaction under nitrogen atmosphere to obtain carbonized magnetic nanoparticles Fe3O4@SiO2 / C, the reaction temperature is above 700°C, and the reaction time is 5h; Step (4): oxidizing the surface of the carbonized magnetic nanoparticles Fe3O4@SiO2 / C by an oxidant to obtain surface-modifiable magnetic nanoparticles FSC, wherein the oxidant is hydrogen peroxide; Step (5): Based on the FSC in step (4), the magnetic nanosensor FSC-D-P0 is prepared by surface modification of ssDNA1 and ssDNA2-P0, and the ratio of aptamer D-P0 to FSC is 0.14:1; specifically, the same molar ssDNA1 and ssDNA2-P0 are mixed with STE buffer, heated in an oil bath at 95°C for 5 minutes, and cooled freely to room temperature to form double-stranded DNA, namely aptamer D-P0, which is a P0 aptamer modified with double-stranded DNA labeled with Cy5; at the same time, the prepared FSC is resuspended in 2.5 mg / mL ultrapure water and the pH is adjusted to 6.0; after adding EDC and NHS, the medium is activated after being vortexed at 25°C for 30 minutes; then, D-P0 is added, and the reaction is continued overnight at 25°C, and the product FSC-D-P0 is purified by multiple magnetic enrichment.

Images