Fluorescence colorimetric sensor for detecting circulating tumor cells and preparation method of fluorescence colorimetric sensor

Abstract

The invention discloses a fluorescent colorimetric sensor for detecting circulating tumor cells and a preparation method of the fluorescent colorimetric sensor. The preparation method comprises the following steps: S4, adding a prepared detection antibody modified with horse radish peroxidase and used for detecting the circulating tumor cells, and incubating; s5, adding a Tris buffer solution and a H2O2 solution, and incubating; s6, preparing an EuMOF nanoflower solution; and S7, adding the EuMOF nanoflower solution and 3, 3 ', 5, 5'-tetramethyl benzidine to react, detecting the fluorescence intensity in the presence of circulating tumor cells with different concentrations by using a luminoscope, detecting the ultraviolet absorbance value in the presence of circulating tumor cells with different concentrations by using a multifunctional microplate reader, and constructing a fluorescence intensity-concentration working curve and an ultraviolet absorbance value-concentration working curve. The method is used for qualitative and quantitative analysis and detection of the circulating tumor cells, realizes bimodal signal response to the circulating tumor cells, and has high sensitivity and simplicity and convenience in operation.

Description

Technical Field

[0001] The present invention belongs to the field of analysis and detection of disease markers using lanthanide metal organic framework materials, and particularly relates to a fluorescence colorimetric sensor for detecting circulating tumor cells and a preparation method thereof. Background Art

[0002] Tumor metastasis, the process by which tumor cells detach from a primary site and seed to distant secondary sites via the circulatory system, forming new tumor foci, is a leading cause of death in cancer patients. Circulating tumor cells (CTCs) are tumor cells that detach from a tumor, invade, and traverse the vasculature. CTCs represent tumor aggressiveness, provide a true reflection of tumor burden, and possess the potential to develop into metastatic lesions. Therefore, as a key target in tumor metastasis research, the development of highly sensitive detection technologies for circulating tumor cells (CTCs) faces significant challenges. Traditional detection methods suffer from complex procedures and insufficient sensitivity. However, CTC levels in peripheral blood are very low, at as low as 1 in every 10⁵ to 10⁷ mononuclear cells. Effectively capturing and detecting circulating tumor cells with diverse phenotypes presents a significant challenge.

[0003] Traditional CTC detection techniques primarily include immunomagnetic capture, density gradient centrifugation, and microfluidic chip technology. However, these methods suffer from high costs, low selectivity and sensitivity, and complex detection processes. Fluorescence and colorimetry dual-modality detection of CTCs offers significant advantages over traditional single-detection methods. First, fluorescence detection offers high sensitivity, enabling accurate detection of circulating tumor cells at low concentrations, enabling early detection, especially when tumor cells are scarce in the blood. Colorimetric detection, on the other hand, relies on a visible color change, avoiding the need for expensive instrumentation, making it simpler to operate and suitable for use in diverse experimental settings. The combination of the high sensitivity of fluorescence detection and the simplicity of colorimetry complement each other, ensuring reliable and accurate test results while also making the detection process more efficient. Compared to single fluorescence or colorimetric detection methods, this dual-modality approach can provide more sensitive and comprehensive detection under diverse experimental conditions. This is because the fluorescence signal rapidly reflects the presence of CTCs, while the colorimetric reaction is simple and intuitive.

[0004] Metal-organic frameworks (MOFs) are a class of porous crystalline nanomaterials constructed through self-assembly of metal nodes and organic ligands. MOFs, with their tunable pore size, large surface area, and high color purity, are widely used in biomarker detection. Their structural composition can be tailored by selecting different metal nodes and organic ligands to suit diverse biomarker detection requirements. Surface functionalization allows MOFs to enhance their selective affinity for target biomarkers by attaching specific organic groups or biomolecules (such as antibodies and aptamers). Lanthanide metal-organic frameworks (Ln-MOFs) offer significant advantages in biomarker detection due to their unique optical and structural properties. Eu-MOFs, due to their unique electronic structure and ionic coordination environment, exhibit excellent red fluorescence. This superior fluorescence provides higher signal intensity, enabling more sensitive and accurate detection, offering unique advantages in the field of fluorescence sensing. Overall, the use of MOFs in biomarker detection represents a promising research direction. Summary of the Invention

[0005] In light of this, the present invention aims to address the shortcomings of existing technologies by providing a fluorescence colorimetric sensor for detecting circulating tumor cells and its preparation method. This fluorescence colorimetric sensor leverages the optical properties of a two-dimensional europium metal-organic framework (EuMOF) material and the synergistic effect of an enzyme-linked immunosorbent assay (ELISA) to achieve a dual-modal signal response to circulating tumor cells, offering high sensitivity and ease of operation.

[0006] The technical solutions adopted are:

[0007] A method for preparing a fluorescent colorimetric sensor for detecting circulating tumor cells of the present invention comprises the following steps:

[0008] S1. Add circulating tumor cell capture antibodies to the ELISA plate;

[0009] S2. Add the prepared bovine serum albumin and incubate;

[0010] S3. Add the prepared circulating tumor cell solution of different concentrations and incubate;

[0011] S4. Add the prepared detection antibody modified with horseradish peroxidase to detect circulating tumor cells and incubate;

[0012] S5. Add Tris buffer solution and H2O2 solution and incubate;

[0013] S6. Ultrasonic dispersion of fluorescent two-dimensional europium metal organic framework material EuMOF nanoflowers in ultrapure water, and oscillation to prepare a EuMOF nanoflower solution;

[0014] S7. EuMOF nanoflower solution and 3,3',5,5'-tetramethylbenzidine were added to react, and the fluorescence intensity in the presence of different concentrations of circulating tumor cells was detected by a fluorescence meter. The ultraviolet absorbance value in the presence of different concentrations of circulating tumor cells was detected by a multifunctional enzyme marker. The fluorescence intensity-concentration working curve and the ultraviolet absorbance value-concentration working curve were constructed, thereby constructing a fluorescent colorimetric sensor.

[0015] Furthermore, in S6, nanoflowers formed by stacking fluorescent two-dimensional europium metal organic framework material EuMOF nanosheets were dispersed in ultrapure water and ultrasonicated for 30 min, and the concentration of the EuMOF nanoflower solution prepared by oscillation was 200 μg / mL.

[0016] Furthermore, in S6, the preparation method of EuMOF nanoflowers comprises the following steps:

[0017] (1) Dissolve 10.0-300.0 mg of europium (III) nitrate hexahydrate in 1-10 mL of N,N-dimethylformamide solvent, sonicate, and vortex.

[0018] (2) Dissolve 10.0-300.0 mg of 2,2':6',2''-terpyridine-4,4',4''-tricarboxylic acid in 1-10 mL of N,N-dimethylformamide solvent, sonicate, and vortex;

[0019] (3) Mix the solutions in step (1) and step (2), add 20-100 μL of glacial acetic acid to adjust the pH of the solution, transfer to a stainless steel reactor lined with polytetrafluoroethylene, and react at 50.0-200.0°C for 6-48 hours;

[0020] (4) A light yellow product was obtained, and N,N-dimethylformamide was washed off by filtration. The product was dried in a vacuum oven at 60-100 °C overnight to obtain EuMOF nanoflowers.

[0021] Furthermore, in S1, 100 μL of 5 μg / mL circulating tumor cell capture antibody was added to a 96-well ELISA plate, incubated at 4°C overnight, and washed three times with PBST to remove the circulating tumor cell capture antibody not fixed on the ELISA plate.

[0022] Furthermore, 100 μL of the prepared 1 vol% bovine serum albumin was added to S2, incubated at 37 °C for 1 h, and washed three times with PBST to remove excess bovine serum albumin.

[0023] Furthermore, 100 μL of the prepared antigen circulating tumor cell solution with different concentrations was added to S3, incubated at 37°C for 1 h, and washed three times with PBST to remove unbound antigen circulating tumor cells.

[0024] Furthermore, in S4, 100 μL of the prepared 20 μg / mL horseradish peroxidase-modified nucleic acid aptamer for detecting circulating tumor cells was added, incubated at 37°C for 1 h, and washed three times with 5 mM, pH = 5.0 Tris buffer solution to remove unbound nucleic acid aptamers.

[0025] Furthermore, 130 μL of 5 mM, pH 5 Tris buffer solution and 10 μL of 10 mM H2O2 solution were added to S5 and incubated at 37°C for 10 min.

[0026] Furthermore, 50 μL of 200 μg / mL EuMOF nanoflower solution and 10 μL of 20 mM 3,3',5,5'-tetramethylbenzidine were added to S7 and reacted at 37 °C for 30 min.

[0027] The fluorescent colorimetric sensor for detecting circulating tumor cells of the present invention is prepared by the above-mentioned preparation method.

[0028] The beneficial technical effects of the present invention are:

[0029] This invention utilizes a horseradish peroxidase-triggered enzyme-linked immunosorbent assay (ELISA) to detect fluorescence intensity and ultraviolet absorbance in the presence of varying concentrations of circulating tumor cells. Fluorescence intensity-concentration and ultraviolet absorbance-concentration working curves are then constructed to produce a fluorescence colorimetric sensor. The fluorescence intensity of circulating tumor cells in a sample is first measured using a fluorimeter, and the ultraviolet absorbance of circulating tumor cells in the sample is then measured using a multifunctional microplate reader. Substituting the values ​​into the working curves allows for quantitative analysis of circulating tumor cells in the sample. This detection method overcomes the shortcomings of existing circulating tumor cell detection technologies, such as the single-method approach. By leveraging the fluorescent properties of the synthesized two-dimensional EuMOF metal-organic framework (OMF), an ELISA-triggered ELISA based on circulating tumor cells is constructed to create a fluorescence colorimetric sensor for qualitative and quantitative analysis of circulating tumor cells. This colorimetric sensor utilizes the synergistic optical properties of the two-dimensional europium metal-organic framework (EuMOF) and the ELISA to achieve a dual-modal signal response to circulating tumor cells, offering high sensitivity and ease of operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram of a fluorescent colorimetric sensor constructed based on EuMOF nanoflowers for detecting circulating tumor cells;

[0031] Figure 2 This is the scanning electron microscope (SEM) characterization image of EuMOF nanoflower;

[0032] Figure 3 Feasibility study diagram for detecting circulating tumor cells at different concentrations using a fluorescence colorimetric sensor based on EuMOF nanoflowers (fluorescence feasibility (a), colorimetric feasibility (b));

[0033] Figure 4 Fluorescence spectra of the fluorescence colorimetric sensor constructed based on EuMOF nanoflowers for detecting circulating tumor cells of different concentrations and the working curve of fluorescence detection of circulating tumor cells (a, b), ultraviolet absorption spectrum and absorbance value working curve of circulating tumor cells (c, d). DETAILED DESCRIPTION

[0034] The present invention is described in detail below through specific examples, but the use and purpose of these exemplary embodiments are only used to illustrate the present invention and do not constitute any form of limitation on the actual protection scope of the present invention, and do not limit the protection scope of the present invention to these.

[0035] Tris-HCl buffer is a tris(hydroxymethyl)aminomethane-hydrochloride buffer solution; PBST, short for phosphate-buffered saline with Tween, is a commonly used biochemical reagent consisting primarily of phosphate buffer with Tween-20 added. TMB is 3,3',5,5'-tetramethylbenzidine.

[0036] The working principle of the fluorescence colorimetric sensor of the present invention is:

[0037] In the present invention, an enzyme-linked immunosorbent assay (ELISA) triggered by horseradish peroxidase is constructed based on circulating tumor cells. 9FU-AS1411@horseradish peroxidase is used as a detection aptamer. When circulating tumor cells are present, horseradish peroxidase converts hydrogen peroxide into hydroxyl radicals, oxidizing TMB to oxTMB, causing the solution to turn blue. At the same time, the ultraviolet spectrum of oxTMB effectively overlaps with the fluorescence excitation-emission spectrum of EuMOF nanoflowers, quenching the fluorescence of EuMOF nanoflowers. The detection principle is as follows: Figure 1 shown.

[0038] According to the presence or absence of circulating tumor cells and the concentration in the system, the corresponding fluorescence intensity and absorbance value are used to analyze and detect circulating tumor cells. Figure 2 shown.

[0039] Example 1

[0040] The preparation method of a fluorescent colorimetric sensor for detecting circulating tumor cell enzymes in this embodiment comprises the following specific steps:

[0041] 1. First, synthesize EuMOF nanoflower materials. The steps are as follows:

[0042] 1) Dissolve 44.606 mg of europium (III) nitrate hexahydrate in 1 mL of N,N-dimethylformamide, sonicate, and vortex.

[0043] 2) Dissolve 36.639 mg of 2,2':6',2''-terpyridine-4,4',4''-tricarboxylic acid in 9 mL of N,N-dimethylformamide, sonicate, and vortex.

[0044] 3) Mix the solutions in step (1) and step (2), add 20 μL of glacial acetic acid to adjust the pH of the solution, transfer to a stainless steel reactor lined with polytetrafluoroethylene, and react at 150 °C for 24 hours;

[0045] 4) The pale yellow product was filtered to remove N,N-dimethylformamide and dried in a vacuum oven at 60°C overnight to obtain EuMOF nanoflowers.

[0046] 2. Add the capture antibody for circulating tumor cells (5 μg / mL, 100 μL) to a 96-well ELISA plate, incubate at 4°C overnight, and wash three times with PBST to remove the capture antibody not fixed to the plate.

[0047] 3. Add the prepared bovine serum albumin (1%, 100 μL), incubate at 37°C for 1 h, and wash three times with PBST to remove excess bovine serum albumin;

[0048] 4. Add circulating tumor cells (100 µL) at different concentrations, incubate at 37°C for 1 h, and wash three times with PBST to remove unbound circulating tumor cells.

[0049] 5. Add the prepared horseradish peroxidase-modified aptamer for detecting circulating tumor cells (20 μg / mL, 100 μL), incubate at 37°C for 1 h, and wash three times with Tris buffer solution (5 mM, pH = 5.0) to remove unbound aptamers.

[0050] 6. Add 130 μL Tris buffer solution (5 mM, pH = 5) and 10 μL H2O2 (10 mM) solution and incubate at 37°C for 10 min.

[0051] 7. Disperse the fluorescent two-dimensional metal organic framework material EuMOF nanosheets into nanoflowers in ultrapure water and sonicate for 30 minutes with oscillation. The concentration of EuMOF material in the solution is 200 μg / mL.

[0052] 8. Add 50 μL of 200 μg / mL EuMOF nanoflower solution and 3,3',5,5'-tetramethylbenzidine (TMB, 20 mM, 10 μL), react at 37°C for 30 min, detect the fluorescence intensity in the presence of different concentrations of circulating tumor cells using a fluorimeter, and detect the ultraviolet absorbance value in the presence of different concentrations of circulating tumor cells using a multifunctional microplate reader, and construct the fluorescence intensity-concentration working curve and the ultraviolet absorbance value-concentration working curve.

[0053] The results showed that when the concentration of circulating tumor cells was in the range of 40-1500 cells / mL, the fluorescence intensity of the system was linearly related to the concentration (FL=-0.0815C+127.73, R 2 = 0.994), the detection limit was 30 cells / mL, and the absorbance value was linearly related to the concentration (Abs = 6.3643*10 -4 C+0.1593, R 2 = 0.998) with a detection limit of 15 cells / mL, as shown in Figure 3.

[0054] The series of detailed descriptions listed above are only specific descriptions of feasible embodiments of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent embodiments or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a fluorescent colorimetric sensor for detecting circulating tumor cells, characterized in that: The steps include: S1. Add circulating tumor cell capture antibodies to the ELISA plate; S2. Add prepared bovine serum albumin and incubate; S3. Add the prepared circulating tumor cell solution of different concentrations and incubate; S4. Add the prepared detection antibody modified with horseradish peroxidase to detect circulating tumor cells and incubate; S5. Add Tris buffer solution and H2O2 solution and incubate; S6. Ultrasonic dispersion of fluorescent two-dimensional europium metal organic framework material EuMOF nanoflowers in ultrapure water, and oscillation to prepare a EuMOF nanoflower solution; S7. EuMOF nanoflower solution and 3,3',5,5'-tetramethylbenzidine were added to react, and the fluorescence intensity in the presence of different concentrations of circulating tumor cells was detected by a fluorescence meter. The ultraviolet absorbance value in the presence of different concentrations of circulating tumor cells was detected by a multifunctional enzyme marker. The fluorescence intensity-concentration working curve and the ultraviolet absorbance value-concentration working curve were constructed, thereby constructing a fluorescent colorimetric sensor.

2. The method for preparing a fluorescent colorimetric sensor for detecting circulating tumor cells according to claim 1, characterized in that: In S6, nanoflowers formed by stacking fluorescent two-dimensional europium metal organic framework material EuMOF nanosheets were dispersed in ultrapure water and ultrasonicated for 30 min, and the concentration of the EuMOF nanoflower solution prepared by oscillation was 200 μg / mL.

3. The method for preparing a fluorescent colorimetric sensor for detecting circulating tumor cells according to claim 1, wherein: In S6, the preparation method of EuMOF nanoflowers comprises the following steps: (1) Dissolve 10.0-300.0 mg of europium (III) nitrate hexahydrate in 1-10 mL of N,N-dimethylformamide solvent, sonicate, and vortex. (2) Dissolve 10.0-300.0 mg of 2,2':6',2''-terpyridine-4,4',4''-tricarboxylic acid in 1-10 mL of N,N-dimethylformamide solvent, sonicate, and vortex; (3) Mix the solutions in step (1) and step (2), add 20-100 μL of glacial acetic acid to adjust the pH of the solution, transfer to a stainless steel reactor lined with polytetrafluoroethylene, and react at 50.0-200.0°C for 6-48 hours; (4) A light yellow product was obtained, and N,N-dimethylformamide was washed off by filtration. The product was dried in a vacuum oven at 60-100 °C overnight to obtain EuMOF nanoflowers.

4. The method for preparing a fluorescent colorimetric sensor for detecting circulating tumor cells according to claim 1, wherein: In S1, 100 μL of 5 μg / mL circulating tumor cell capture antibody was added to a 96-well ELISA plate, incubated at 4°C overnight, and washed three times with PBST to remove the circulating tumor cell capture antibody not fixed on the ELISA plate.

5. The method for preparing a fluorescent colorimetric sensor for detecting circulating tumor cells according to claim 4, characterized in that: To S2, 100 μL of the prepared 1 vol% bovine serum albumin was added, incubated at 37°C for 1 h, and washed three times with PBST to remove excess bovine serum albumin.

6. The method for preparing a fluorescent colorimetric sensor for detecting circulating tumor cells according to claim 5, characterized in that: To S3, 100 μL of the prepared antigen circulating tumor cell solution with different concentrations was added, incubated at 37°C for 1 h, and washed three times with PBST to remove unbound antigen circulating tumor cells.

7. The method for preparing a fluorescent colorimetric sensor for detecting circulating tumor cells according to claim 6, characterized in that: To S4, 100 μL of the prepared 20 μg / mL horseradish peroxidase-modified nucleic acid aptamer for detecting circulating tumor cells was added, incubated at 37°C for 1 h, and washed three times with 5 mM Tris buffer solution, pH = 5.0, to remove unbound nucleic acid aptamers.

8. The method for preparing a fluorescent colorimetric sensor for detecting circulating tumor cells according to claim 7, characterized in that: To S5, add 130 μL of 5 mM, pH 5 Tris buffer solution and 10 μL of 10 mM H2O2 solution and incubate at 37°C for 10 min.

9. The method for preparing a fluorescent colorimetric sensor for detecting circulating tumor cells according to claim 7, wherein: To S7, 50 μL of 200 μg / mL EuMOF nanoflower solution and 10 μL of 20 mM 3,3',5,5'-tetramethylbenzidine were added and reacted at 37°C for 30 min.

10. A fluorescent colorimetric sensor for detecting circulating tumor cells, which is prepared by the preparation method according to any one of claims 1 to 8.

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