Probe and detection method for detecting CTC (circulating tumor cell) by bispecific electrochemical sensor system

Abstract

The invention provides a probe for detecting CTC (circulating tumor cell) by a bispecific electrochemical sensor system and a detection method, and belongs to the technical field of biological detection. The detection system provided by the invention comprises an EpCAM / MUC1 aptamer bispecific coupling magnetic bead CTC capture probe and an EpCAM / MUC1 aptamer bispecific coupling nano-enzyme CTC signal probe. According to the detection method, the capture efficiency is improved by utilizing the synergistic effect of the two aptamers. According to the detection method, the peripheral blood circulating tumor cells can be rapidly detected under the condition that the sample size is small. The detection method is good in accuracy and high in sensitivity, and can provide accurate detection results for disease diagnosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a probe and a detection method for detecting CTCs by using a dual-specificity electrochemical sensor system. BACKGROUND

[0002] The prevention, detection and treatment of cancer are still major medical problems in the world. In the progression of cancer, blood-borne cancer cells, i.e. peripheral blood circulating tumor cells (CTCs), are one of the important factors and markers of tumor metastasis and deterioration. CTCs are detached from solid tumor lesions into peripheral blood circulation through spontaneous or diagnostic and therapeutic operations, most of which undergo apoptosis or are phagocytosed in peripheral blood, and only a small number of them can escape and anchor to develop into metastatic lesions. Clinical studies have shown that the number and molecular characteristics of CTCs are significantly related to the prognosis and survival period of patients, and dynamic monitoring can evaluate the treatment effect and guide individualized treatment, thereby providing an important intervention window for blocking the metastasis chain and improving the quality of life of patients.

[0003] Among more than 50 CTC detection technologies, CellSearch is the only FDA-approved CTC detection method. This system detects epithelial-derived cells by EpCAM antibody magnetic bead enrichment, which has significant limitations: it can only recognize typical epithelial cells expressing EpCAM, and is prone to miss cells undergoing epithelial-mesenchymal transition (EMT), non-EpCAM tumor cells and cell clusters. In addition, free normal epithelial cells caused by inflammation can cause false positives, especially in tumors such as liver cancer with low EpCAM expression, which has insufficient sensitivity. Other detection methods based on surface antigens have poor specificity due to the diversity of tumor antigens and the overlap with normal cells, and no related kit has been approved in China. In addition, CTC detection faces the problem of rarity, and 10 mL of blood contains only a few to dozens of CTCs, which need to be accurately captured from billions of blood cells. The existing "enrichment first and identification later" strategy generally has a cumbersome enrichment step, which leads to cell loss, and the immunofluorescence staining method is prone to false positives and has insufficient sensitivity. Therefore, it is an urgent need in the field to develop a new detection technology that has broad-spectrum recognition ability, high specificity and can preserve cell activity. SUMMARY

[0004] The purpose of the present application is to provide a probe and a detection method for detecting CTCs by using a dual-specificity electrochemical sensor system.

[0005] The present application provides a signal probe CMA, which is a nanoparticle obtained by grafting MUC1 aptamer and EpCAM aptamer on the surface of CAT@Fe(SS)MOF; The CAT@Fe(SS)MOF is prepared from ferric chloride hexahydrate, dithiodiglycolic acid, catalase, polyvinylpyrrolidone and water; The nucleotide sequence of the MUC1 aptamer is shown as SEQ ID NO. 1; and the nucleotide sequence of the EpCAM aptamer is shown as SEQ ID NO. 2.

[0006] Further, the preparation method of the CAT@Fe(SS) MOF comprises the following steps: The aqueous solution of ferric chloride hexahydrate is poured into the aqueous solution of dithio-diacetic acid, and then the aqueous solution of catalase and the aqueous solution of polyvinylpyrrolidone are sequentially added to react, to obtain the CAT@Fe(SS) MOF; The volume ratio of the aqueous solution of ferric chloride hexahydrate, the aqueous solution of dithio-diacetic acid, the aqueous solution of catalase and the aqueous solution of polyvinylpyrrolidone is 1:(1-5):(0.1-0.5):(1-5); And / or, the concentration of the aqueous solution of ferric chloride hexahydrate is 5-10 mg / mL; And / or, the concentration of the aqueous solution of dithio-diacetic acid is 5-10 mg / mL; And / or, the concentration of the aqueous solution of catalase is 1-5 mg / mL; And / or, the concentration of the aqueous solution of polyvinylpyrrolidone is 10-15 mg / mL; And / or, the temperature of the reaction is 20-40℃, and the reaction time is 10-30 min.

[0007] Preferably, The volume ratio of the aqueous solution of ferric chloride hexahydrate, the aqueous solution of dithio-diacetic acid, the aqueous solution of catalase and the aqueous solution of polyvinylpyrrolidone is 1:2:0.2:1.2; The concentration of the aqueous solution of ferric chloride hexahydrate is 10 mg / mL; And / or, the concentration of the aqueous solution of dithio-diacetic acid is 5.4 mg / mL; And / or, the concentration of the aqueous solution of catalase is 1 mg / mL; And / or, the concentration of the aqueous solution of polyvinylpyrrolidone is 13.3 mg / mL; And / or, the temperature of the reaction is 25℃, and the reaction time is 10 min.

[0008] The application further provides a preparation method of the signal probe CMA, which comprises the following steps: (a) adding the CAT@Fe(SS) MOF dispersion liquid into the carboxyl activation liquid and uniformly mixing to obtain a mixed liquid; (b) adding the MUC1 aptamer solution into the mixed liquid and incubating; (c) adding the EpCAM aptamer solution into the mixed liquid after the incubation of step (b) and incubating; (d) centrifuging the product after incubation, washing, drying, and obtaining.

[0009] Further, In step (a), the carboxyl activation solution is a PBS solution containing EDC and NHS. And / or, in step (a), the volume ratio of the CAT@Fe(SS) MOF dispersion solution and the carboxyl activation solution is 1: (1-5). And / or, in step (a), the concentration of the CAT@Fe(SS) MOF dispersion solution is 1-5 mg / mL. And / or, in step (b), the volume ratio of the mixed solution and the MUC1 aptamer solution is (1-3): 1. And / or, in step (b), the concentration of the MUC1 aptamer solution is 100-300 μM. And / or, in step (b), the temperature of the incubation is 35-37 ℃, and the incubation time is 20-30 h. And / or, in step (c), the volume ratio of the EpCAM aptamer solution and the mixed solution in step (b) is 1: (1-3). And / or, in step (c), the concentration of the EpCAM aptamer solution is 100-300 μM. And / or, in step (c), the temperature of the incubation is 35-37 ℃, and the incubation time is 20-30 h.

[0010] Preferably, In step (a), the concentration of EDC in the carboxyl activation solution is 8-9 mg / mL, and the concentration of NHS is 5-6 mg / mL. And / or, in step (a), the volume ratio of the CAT@Fe(SS) MOF dispersion solution and the carboxyl activation solution is 1:4. And / or, in step (a), the concentration of the CAT@Fe(SS) MOF dispersion solution is 4 mg / mL. And / or, in step (b), the concentration of the MUC1 aptamer solution is 100 μM. And / or, in step (b), the temperature of the incubation is 37 ℃, and the incubation time is 24 h. And / or, in step (c), the concentration of the EpCAM aptamer solution is 100 μM. And / or, in step (c), the temperature of the incubation is 37 ℃, and the incubation time is 24 h.

[0011] More preferably, Both the MUC1 aptamer and the EpCAM aptamer are connected to an amino group at the 5' end of the above sequence.

[0012] The application further provides a capture probe FA, which is a nanoparticle obtained by grafting a MUC1 aptamer and an EpCAM aptamer on the surface of an amino-bearing Fe3O4 nanoparticle. The nucleotide sequence of the MUC1 aptamer is shown as SEQ ID NO. 1; and the nucleotide sequence of the EpCAM aptamer is shown as SEQ ID NO. 2. The preparation method of the amino-bearing Fe3O4 nanoparticle comprises the following steps: dissolving ferric chloride hexahydrate and anhydrous sodium acetate in ethylene glycol, and then adding hexanediamine to obtain a mixed solution; and reacting the mixed solution to obtain the amino-bearing Fe3O4 nanoparticle.

[0013] Further, the mass-volume ratio of the ferric chloride hexahydrate, the anhydrous sodium acetate, the ethylene glycol and the hexanediamine is 1g: (4-5) g: (30-50) mL: (1-5) mL. And / or, the reaction temperature is 200-300 DEG C, and the reaction time is 5-10 h.

[0014] Preferably, The mass-volume ratio of the ferric chloride hexahydrate, the anhydrous sodium acetate, the ethylene glycol and the hexanediamine is 1g: 4g: 30mL: 3.6mL. And / or, the reaction temperature is 200 DEG C, and the reaction time is 6 h.

[0015] The application further provides a preparation method of the capture probe FA, which comprises the following steps: (A) adding MUC1 aptamer solution and EpCAM aptamer solution into a carboxyl activation solution respectively to activate the carboxyl groups of the aptamers; (B) adding the MUC1 aptamer solution after activation of the carboxyl groups into a Fe3O4 nanoparticle dispersion solution for incubation; (C) adding the EpCAM aptamer solution for incubation after the incubation of step (B); (D) centrifuging, washing and drying the product after the incubation to obtain the product.

[0016] Further, In step (A), the carboxyl activation solution is a PBS solution containing EDC and NHS; And / or, in step (A), the volume ratio of the MUC1 aptamer solution to the carboxyl activation solution is 1: (1-3); And / or, in step (A), the concentration of the MUC1 aptamer solution is 100-300 muM; And / or, in step (A), the volume ratio of the EpCAM aptamer solution to the carboxyl activation solution is 1: (1-3); And / or, in step (A), the concentration of the EpCAM aptamer solution is 100~300 μM; And / or, in step (B), the volume ratio of the MUC1 aptamer solution to the Fe3O4 nanoparticle dispersion is 1:(1~3). And / or, in step (B), the concentration of the Fe3O4 nanoparticle dispersion is 10~15 mg / mL; And / or, in step (B), the incubation temperature is 35~37 ℃ and the incubation time is 20~30 h; And / or, in step (C), the volume ratio of the EpCAM aptamer solution to the Fe3O4 nanoparticle dispersion is 1:(1~3). And / or, in step (C), the incubation temperature is 35~37 ℃ and the incubation time is 20~30 h.

[0017] Preferably, In step (A), the concentration of EDC in the carboxyl activating solution is 6~7 mg / mL, and the concentration of NHS is 4~5 mg / mL; And / or, in step (A), the concentration of the MUC1 aptamer solution is 100 μM; And / or, in step (A), the concentration of the EpCAM aptamer solution is 100 μM; And / or, in step (B), the concentration of the Fe3O4 nanoparticle dispersion is 10 mg / mL; And / or, in step (B), the incubation temperature is 37 °C and the incubation time is 24 h; And / or, in step (C), the incubation temperature is 37 °C and the incubation time is 24 h.

[0018] This invention also provides a method for detecting CTCs based on a dual-specificity electrochemical sensor system, which includes the following steps: (1) The solution of the aforementioned signal probe CMA and the solution of the aforementioned capture probe FA were incubated with human blood samples to obtain CMA-CTC-FA; (2) Using magnets to enrich CMA-CTC-FA; (3) The enriched CMA-CTC-FA was added to a solution containing H2O2 and TMB to react and obtain a CMA-CTC-FA solution; (4) The electrical signal of the CMA-CTC-FA solution was detected by differential pulse voltammetry, and the number of CTCs was calculated based on the standard curve.

[0019] Furthermore, In step (1), the concentration of signal probe CMA in the solution is 0.2~2 mg / mL, and the concentration of capture probe FA in the solution is 0.5~2 mg / mL; And / or, in step (1), the solvent in the solution of the signal probe CMA and the solution of the capture probe FA is PBS; And / or, in step (1), the volume ratio of the solution of the signal probe CMA, the solution of the capture probe FA, and human blood is (0.5~0.6):(0.1~0.3):1; And / or, in step (1), the incubation temperature is 35~37℃ and the incubation time is 20~70min; And / or, in step (3), the concentration of H2O2 in the solution is 20~30mM and the concentration of TMB is 10~20mM; And / or, in step (3), the solvent of the solution is PBS, and the pH value of PBS is 4~10; And / or, in step (3), the reaction temperature is 20~40℃ and the reaction time is 1~5h; And / or, in step (4), the voltage detected by the differential pulse voltammetry is 0.2~0.4V, the pulse amplitude is 50~100 mV, and the pulse width is 50~100 ms; And / or, in step (4), the standard curve is y = 0.9928x + 9.3364, R 2 =0.9971; x is the number of cells, and y is the DPV signal value.

[0020] Preferably, In step (1), the concentration of signal probe CMA in the solution is 2 mg / mL, and the concentration of capture probe FA in the solution is 2 mg / mL. And / or, in step (1), the pH of the PBS is 7.4; And / or, in step (1), the volume ratio of the solution of the signal probe CMA, the solution of the capture probe FA, and human blood is 0.6:0.1:1; In actual use, 1 mL of human blood is typically taken, along with 600 μL of the CMA signal probe solution and 100 μL of the FA capture probe solution.

[0021] And / or, in step (1), the incubation temperature is 37°C and the incubation time is 30 min; And / or, in step (3), the concentration of H2O2 in the solution is 20 mM and the concentration of TMB is 11 mM; And / or, in step (3), the pH value of the PBS is 8; And / or, in step (3), the reaction temperature is 25~30℃ and the reaction time is 1h; And / or, in step (4), the voltage detected by the differential pulse voltammetry is 0.2~0.4V, the pulse amplitude is 50mV, and the pulse width is 50ms.

[0022] This invention employs a mild Fe-MOF in-situ encapsulation strategy for CAT synthesis, successfully preparing a POD-like nanozyme with a synergistic enhancement effect between the natural enzyme and the synthase. Simultaneously, by grafting MUC1 and EpCAM aptamers onto the surfaces of the nanozyme and magnetic nanoparticles via amidation, these aptamers are used as signal probes and capture probes, respectively, successfully constructing a bispecific electrochemical biosensor for recognizing circulating tumor cells (CTCs) in peripheral blood. Specifically, the system of this invention exhibits good sensitivity and specificity in detecting A549 non-small cell lung cancer cells (CTCs).

[0023] The present invention has achieved the following beneficial effects: (1) The synergistic catalytic effect of Fe-MOF in situ encapsulation of CAT can amplify the electrochemical signal, and combined with magnetic enrichment technology, the target cells can be accurately separated with only 1 mL of whole blood. (2) High-efficiency enrichment and detection of CTCs with low to medium abundance can be achieved, avoiding cell loss caused by sample size limitations in traditional methods. At the same time, the high specificity of aptamer recognition effectively eliminates interference from blood cells and reduces false positives, making it particularly suitable for clinical dynamic monitoring scenarios that require repeated sampling; (3) Based on the advantages of electrochemical detection, this system uses the stable redox current generated by the decomposition of H2O2 by POD-like nanozymes as the signal output. Its dual-enzyme cascade effect significantly improves the sensitivity and can detect CTCs as low as 2 cells / mL.

[0024] (4) The electrochemical platform effectively distinguishes CTCs from normal epithelial cells through the dual specificity of nanozyme catalysis and aptamer recognition. Combined with magnetic probe for rapid separation and signal amplification, it achieves the integration of "enrichment-detection", shortening the detection time to 95 minutes.

[0025] (5) The miniaturization of electrochemical sensing devices facilitates integration with portable instruments, enabling bedside instant detection, especially meeting the needs of primary healthcare scenarios.

[0026] In summary, this invention provides a method for detecting circulating tumor cells (CTCs) using a dual-specificity electrochemical sensor system. The detection system comprises an EpCAM / MUC1 aptamer-coupled dual-specificity magnetic bead CTC capture probe and an EpCAM / MUC1 aptamer-coupled dual-specificity nanozyme CTC signal probe. This detection method utilizes the synergistic effect of the dual aptamers to enhance capture efficiency. This method enables rapid detection of circulating tumor cells in peripheral blood with small sample sizes. Furthermore, this method offers high accuracy and sensitivity, providing accurate results for disease diagnosis.

[0027] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0028] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the synthesis of CMA nanoparticles according to the present invention.

[0030] Figure 2 The images show SEM images of the Fe(SS)MOF, CAT@Fe(SS)MOF, and CMA nanoparticles synthesized in this invention.

[0031] Figure 3 This is a TEM image of the CMA nanoparticles synthesized in this invention.

[0032] Figure 4 The graph shows the peroxidase (POD) activity detection results of CAT and the Fe(SS)MOF, CAT@Fe(SS)MOF, and CMA nanoparticles synthesized in this invention.

[0033] Figure 5 This is a DPV curve of the CMA nanoparticles synthesized in this invention.

[0034] Figure 6 This is a TEM image of the FA nanoparticles synthesized in this invention.

[0035] Figure 7 FT-IR images of CAT, Fe3O4, FA nanoparticles, Fe(SS)MOF, CAT@Fe(SS)MOF, and CMA nanoparticles.

[0036] Figure 8 This is a schematic diagram of the electrochemical detection process for CTC.

[0037] Figure 9 This is a graph showing the linear relationship between the number of tumor cells and the DPV signal in PBS buffer solution using the CMA-FA detection system of this invention.

[0038] Figure 10 Figure showing the results of optimized parameters for CTC detection using the CMA-FA electrochemical cell sensor.

[0039] Figure 11 The results of the CMA-FA electrochemical cell sensor detection method of the present invention in detecting healthy individuals and lung cancer patients are shown in the following figures: a) Detection results of the CMA-FA electrochemical cell sensor detection method on clinical specimens of healthy individuals and lung cancer patients; b) ROC curve results of the CMA-FA electrochemical cell sensor detection method in distinguishing between healthy individuals and lung cancer patients.

[0040] Figure 12 The following figures show the results of the cell detection specificity analysis of the CMA-FA electrochemical cell sensor detection method of the present invention: a is a comparison of the expression of different cell surface antigens; b is a DPV signal diagram of different cells acting on the CMA-FA detection system. Detailed Implementation

[0041] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0042] Unless otherwise specified, the solvent in the solutions of this invention is water.

[0043] Example 1: Preparation of the signal probe CMA The bispecific signal probe is a CMA nanoparticle, and the synthesis process is as follows: Figure 1 As shown, it is prepared according to the following steps: (1) Preparation of Fe(SS)MOF: 5 mL of ferric chloride hexahydrate solution (concentration of ferric chloride hexahydrate was 10 mg / mL) was slowly poured into 10 mL of dithiodiacetic acid solution (concentration of dithiodiacetic acid was 5.4 mg / mL). Then, 6 mL of polyvinylpyrrolidone (PVP) solution (concentration of PVP was 13.3 mg / mL) and 1 mL of water were added. The mixture was stirred at room temperature (25℃) for 10 min, and the product was milky yellow. The reaction product was centrifuged (centrifugation conditions were 13,750 × g, 15 min), and the precipitate was washed twice with water to obtain Fe(SS)MOF with carboxyl groups on the surface, which was stored in 5 mL of water for later use.

[0044] (2) Preparation of CAT@Fe(SS)MOF: 5 mL of ferric chloride hexahydrate solution (concentration of ferric chloride hexahydrate was 10 mg / mL) was slowly poured into 10 mL of dithiodiacetic acid solution (concentration of dithiodiacetic acid was 5.4 mg / mL). Then, 1 mL of catalase (CAT) solution (CAT concentration was 1 mg / mL) and 6 mL of polyvinylpyrrolidone (PVP) solution (PVP concentration was 13.3 mg / mL) were added sequentially. The mixture was stirred at room temperature (25℃) for 10 min, and the product was milky yellow. The reaction product was centrifuged (centrifugation conditions: 13,750 × g, 15 min), and the precipitate was washed twice with water to obtain CAT@Fe(SS)MOF with carboxyl groups on the surface and CAT encapsulated inside. The precipitate was stored in 5 mL of water for later use.

[0045] (3) Preparation of carboxyl activation solution: 65 mg EDC and 40 mg NHS were added to 8 mL PBS solution and stirred to dissolve to obtain carboxyl activation solution.

[0046] (4) Preparation of CMA nanoparticles: 2 mL of CAT@Fe(SS)MOF dispersion (CAT@Fe(SS)MOF concentration was 4 mg / mL) was poured into 8 mL of carboxyl activation solution. 530 μL of the above mixture was taken, and then 400 μL of MUC1-NH2 aptamer solution (100 μM) was added. The mixture was incubated in a constant temperature shaker at 37 ℃ for 24 h. Then, 210 μL of EpCAM-NH2 aptamer solution (100 μM) was added, and the mixture was incubated for another 24 h. The incubated product was then centrifuged (centrifugation conditions: 13,750 × g, 15 min). The precipitate was washed twice with water and vacuum dried to remove moisture, yielding the final product (signal probe—CMA nanoparticles). The particle size of the CMA nanoparticles was 100 nm to 200 nm. MUC1-NH2 aptamer and EpCAM-NH2 aptamer coated the surface of the Fe(SS)MOF material. MUC1-NH2 aptamers and EpCAM-NH2 aptamers can be grafted onto the surface of CTC cells via amidation.

[0047] MUC1 aptamer sequence (5' to 3'): GCA GTT GAT CCT TTG GAT ACC CTG G (SEQ ID NO.1) EpCAM aptamer sequence (5' to 3'): CAC TAC AGA GGT TGC GGTC TGT CCC ACG TTGTCA TGG GGG GTT GGC CTG (SEQ ID NO.2) Both the MUC1-NH2 aptamer and the EpCAM-NH2 aptamer have an amino group attached to the 5' end of the above sequence.

[0048] The surface of the aforementioned CMA nanoparticles is co-modified with specific aptamers targeting MUC1 and EpCAM. Utilizing their high affinity and selective binding properties, dual-target synergistic recognition of tumor cells can be achieved to enrich and identify targeted tumor cells with high sensitivity.

[0049] SEM images of Fe(SS)MOF, CAT@Fe(SS)MOF, and CMA nanoparticles, such as... Figure 2 As shown, by Figure 2 It can be seen that the SEM images of the products at each stage show that the Fe(SS)MOF morphology before and after CAT encapsulation is uniform spheres with no significant difference in size (about 30 nm), indicating that Fe(SS)MOF can maintain excellent structural stability under the synthesis conditions.

[0050] TEM images of CMA nanoparticles Figure 3 As shown, by Figure 3 As can be seen, the TEM image of CMA clearly shows its core-shell structure, especially the layered structure of Fe and S elements in its shell, which further verifies that CAT is encapsulated inside Fe(SS)MOF. The N element distribution map in the transmission electron microscope shows that the aminated MUC1 / EpCAM was successfully coupled to the CAT@Fe(SS)MOF surface.

[0051] Peroxidase (POD) activity was analyzed using a TMB-H2O2 colorimetric system. Specifically, 100 μL of acetate-sodium acetate buffer dispersions (1 mg / mL) of CAT, Fe(SS)MOF, CAT@Fe(SS)MOF, and CMA were added dropwise to each well of a 96-well plate. 100 μL of ready-to-use TMB solution was added, and the plates were incubated at room temperature in the dark for 1 h. The OD value at 650 nm was recorded using a microplate reader. The peroxidase (POD) activity detection results for CAT, synthesized Fe(SS)MOF, CAT@Fe(SS)MOF, and CMA nanoparticles are shown below. Figure 4 As shown. By Figure 4 It can be seen that the POD activity of Fe(SS)MOF is significantly higher than that of natural CAT, while the activity of CAT@Fe(SS)MOF is further enhanced due to synergistic catalysis. Although the POD activity of CMA nanoparticles is lower than that of CAT@Fe(SS)MOF, it is still significantly higher than that of natural CAT.

[0052] The specific method for differential pulse voltammetry (DPV) detection is as follows: 600 μL of CMA signal probe solution (CMA signal probe concentration in the solution is 2 mg / mL, solvent is PBS, pH=7.4) and 100 μL of FA capture probe solution prepared in Example 2 (FA capture probe concentration in the solution is 2 mg / mL, solvent is PBS, pH=7.4) are added together to 1 mL of cell density 1×10⁻⁶ cells / mL. 4 Suspensions of various tumor cells (A549, Caco-2, SNU719, Ges-1, and RAW264.7) were collected at cell / mL. The incubation volume was increased to 2 mL with PBS (pH=7.4) and incubated at 37°C for 30 min. The target cells bound to the probe were enriched by adsorption with a strong magnet for 5 min. The cells were washed twice with PBS buffer (pH=7.4). The enriched reaction product was dispersed in 255 μL of PBS solution (pH=8) containing H2O2 (35 μL, 20 mM) and TMB (20 μL, 11 mM) and reacted at room temperature in the dark for 1 h to obtain CMA-cell-FA solution. 100 μL of CMA-cell-FA solution was uniformly dropped onto a screen-printed electrode. The obtained electrical signal was detected by differential pulse voltammetry (DPV) in the range of 0.2~0.4 V, with a pulse amplitude of 50 mV and a pulse width of 50 ms. The DPV curves of the synthesized CMA nanoparticles are as follows: Figure 5 As shown. By Figure 5 It can be seen that CMA only shows a strong DPV signal peak near 0.3 eV when coexisting with TMB-H2O2, demonstrating its high signal-to-noise ratio potential as an electrochemical signal probe for specific identification of CTC.

[0053] Example 2: Preparation of the capture probe FA (1) Preparation of Fe3O4: 1 g of ferric chloride hexahydrate, 4 g of anhydrous sodium acetate, and 30 mL of ethylene glycol were heated and stirred at 70 °C until completely dissolved. Then, 3.6 mL of hexamethylenediamine was added and stirring continued for 20 min. The mixture was placed in a polytetrafluoroethylene reactor and reacted in a high-temperature (200 °C) oven for 6 h. After the reaction was completed, the reactants were washed twice with ethanol and twice with pure water to effectively remove the solvent and unbound hexamethylenediamine. In each rinsing step, the nanoparticles were separated from the supernatant using magnetic force. After vacuum drying, Fe3O4 with amino groups on the surface was obtained.

[0054] (2) Preparation of FA nanoparticles: 10 mL of PBS carboxyl activation solution containing 65 mg EDC and 40 mg NHS was prepared. 400 μL of the carboxyl activation solution was added to 210 μL of MUC1 aptamer solution (MUC1 aptamer concentration of 100 μM) and 210 μL of EpCAM aptamer solution (EpCAM aptamer concentration of 100 μM) to activate the carboxyl groups of the aptamers. 400 μL of the activated MUC1 aptamer was added to 530 μL of Fe3O4 dispersion (Fe3O4 concentration of 10 mg / mL) and incubated in a constant temperature shaker at 37 ℃ for 24 h. Then, 210 μL of EpCAM aptamer was added and incubated for another 24 h. After incubation, the nanoparticles were washed twice with pure water and dried under vacuum to remove moisture, yielding the final product, FA nanoparticles.

[0055] The nucleotide sequence of the MUC1 aptamer is shown in SEQ ID NO.1; the nucleotide sequence of the EpCAM aptamer is shown in SEQ ID NO.2.

[0056] The FA nanoparticles prepared in this embodiment have super magnetic properties, and the MUC1 aptamer and EpCAM aptamer are combined with the FA nanoparticles through an amide reaction.

[0057] The FA nanoparticles prepared in this embodiment are TEM-like. Figure 6 As shown, by Figure 6 It can be seen that the transmission electron microscopy characterization of FA shows that the signal intensity distribution of all elements highly coincides with the morphological profile of the nanofiber. The distribution map and content analysis of the C element unique to its aptamer also preliminarily prove that the carboxylated MUC1 / EpCAM was successfully coupled to the Fe3O4 surface.

[0058] FT-IR of CAT, Fe3O4, FA nanoparticles, Fe(SS)MOF, CAT@Fe(SS)MOF, and CMA nanoparticles during synthesis, such as... Figure 7 As shown, by Figure 7 Infrared spectroscopy analysis revealed an enhanced CAT carboxyl peak at 1400 cm⁻¹ and a newly added amide III band at 1285 cm⁻¹ in CAT@Fe(SS)MOF, confirming that CAT is encapsulated within the MOF (consistent with TEM results). The newly appearing peaks at 3574 cm⁻¹ (Fe-OH) and 650 cm⁻¹ (Fe-O) support the nucleation mechanism of CAT through coordination with Fe³⁺ via negatively charged groups. After aptamer modification, CMA exhibited a characteristic C=O peak at 1638 cm⁻¹; FA showed a -CONH- peak at 1574 / 1647 cm⁻¹, verifying successful grafting of the aptamer via amidation. These results collectively confirm the stepwise assembly process of the material.

[0059] Example 3: Establishment of a CTC enrichment and detection system based on CMA-FA I. Construction of the Standard Curve A schematic diagram of the detection process is shown below. Figure 8 As shown. The linear relationship between cell number and DPV signal was established by following these steps: (1) 600 μL of CMA signal probe solution (the concentration of CMA signal probe in the solution is 2 mg / mL, the solvent is PBS, pH 7.4) and 100 μL of FA capture probe solution (the concentration of FA capture probe in the solution is 2 mg / mL, the solvent is PBS, pH=7.4) were added to 1 mL of cell solution containing different numbers of CTC cells (A549 cells). The incubation volume was increased to 2 mL with PBS (pH=7.4), and the mixture was incubated at 37℃ for 30 min to obtain CMA-A549-FA.

[0060] (2) CMA-A549-FA was separated by adsorption with a strong magnet and washed twice with PBS (pH=7.4).

[0061] (3) The enriched reactants were dispersed in 255 μL of PBS solution (pH=8) containing H2O2 (35 μL, 20 mM) and TMB (20 μL, 11 mM) and reacted at room temperature in the dark for 1 h to obtain CMA-cell-FA solution.

[0062] (4) A specific volume (100 μL) of CMA-A549-FA solution was dropped onto a screen-printed electrode, and the obtained electrical signal was detected using differential pulse voltammetry (DPV) in the range of 0.2–0.4 V, with a pulse amplitude of 50 mV and a pulse width of 50 ms. The DPV signal intensity for different cell numbers was obtained. The linear relationship between the DPV signal and the cell number was calculated. The obtained linear relationship between the DPV signal and the cell number is as follows: Figure 9 As shown. By Figure 9 It can be seen that the limit of detection (LoD) of this detection method is 2 cells / mL, which is lower than the conventional clinical positive threshold. Within the detection range of 2-50 cells / mL, it ensures excellent linear response characteristics in the clinically relevant concentration range, y=0.9228x+9.3364, R0. 2 =0.9971. Therefore, the detection method of the present invention has high sensitivity. Furthermore, the detection method only takes 95 minutes, making it fast.

[0063] II. Detection of circulating tumor cells (CTCs) in peripheral blood The method for detecting circulating tumor cells in peripheral blood using the CMA-FA dual-specificity electrochemical sensor system is as follows: (1) Collect 1 mL of human blood, add 600 μL of CMA signal probe solution (the concentration of CMA signal probe in the solution is 2 mg / mL, the solvent is PBS, pH=7.4) and 100 μL of FA capture probe solution (the concentration of FA capture probe in the solution is 2 mg / mL, the solvent is PBS, pH=7.4) to the human blood at the same time, and add PBS (pH=7.4) to make up the final incubation volume to 2 mL. Incubate at 37℃ for 30 min to obtain CMA-CTC-FA.

[0064] (2) Use a strong magnet to separate CMA-CTC-FA and wash twice with PBS (pH=7.4).

[0065] (3) The enriched reactants were dispersed in 255 μL of PBS (pH=8) containing H2O2 (35 μL, 20 mM) and TMB (20 μL, 11 mM) and reacted at room temperature in the dark for 1 h to obtain CMA-CTC-FA solution.

[0066] (4) 100 μL of CMA-CTC-FA solution was dropped onto the screen-printed electrode. The obtained electrical signal was detected using differential pulse voltammetry (DPV) in the range of 0.2~0.4 V, with a pulse amplitude of 50 mV and a pulse width of 50 ms. Substituting these values ​​into the linear relationship formula between the DPV signal and the number of electrons (y=0.9228x+9.3364, R...), the signal was analyzed. 2 =0.9971), thus obtaining the number of CTCs.

[0067] The detection method of this invention only requires 1 mL of peripheral blood sample to complete the detection.

[0068] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0069] Experiment 1: Optimization Experiment of CTC Detection Conditions A CTC detection system was established according to the method described in Example 3, using A549 cells as a model (1×10⁻⁶). 4 / mL), optimizing the concentration of the signal probe, the pH during the reaction of CMA-CTC-FA with TMB and H2O2, and the incubation time of cells and aptamers, respectively. The results showed ( Figure 10 The DPV signal intensity reached its peak under optimal conditions (CMA solution: 600 μL, CMA concentration: 2 mg / mL; FA solution: 100 μL, FA concentration: 2 mg / mL; reaction pH = 8; incubation: 30 min). The signal intensity initially increased and then decreased within other parameter ranges, indicating that the system has high sensitivity and controllable conditions.

[0070] 1. The specific method for optimizing the concentration of the signal probe is as follows: (1) Add 200, 400, 600, 800, and 1000 μL of CMA signal probe solution (CMA signal probe concentration in the solution is 2 mg / mL, solvent is PBS, pH=7.4) and 100 μL of FA capture probe solution (FA capture probe concentration in the solution is 2 mg / mL, solvent is PBS, pH=7.4) to 1 mL of A549 cells containing 1×10⁻⁶ cells. 4 The cell solution was prepared at / mL, and the incubation volume was increased to 2 mL with PBS (pH=7.4). The mixture was then incubated at 37℃ for 1 h to obtain CMA-A549-FA.

[0071] (2) CMA-A549-FA was separated by adsorption with a strong magnet and washed twice with PBS (pH=7.4).

[0072] (3) The enriched reactants were dispersed in 255 μL of PBS (pH=7.4) containing H2O2 (35 μL, 20 mM) and TMB (20 μL, 11 mM) and reacted at room temperature in the dark for 1 h to obtain CMA-A549-FA solution.

[0073] (4) 100 μL of CMA-A549-FA solution was dropped onto the screen-printed electrode. The obtained electrical signal was detected using differential pulse voltammetry (DPV) in the range of 0.2~0.4 V, with a pulse amplitude of 50 mV and a pulse width of 50 ms. Substituting these values ​​into the linear relationship formula between the DPV signal and the number of pulses (y=0.9228x+9.3364, R...), the signal was analyzed. 2 =0.9971), the DPV signal intensity under this method with different signal probe concentrations was obtained. The results are as follows. Figure 10 As shown, a volume of 600 μL is optimal.

[0074] 2. The specific method for optimizing the pH during the reaction of CMA-CTC-FA with TMB and H2O2 is as follows: (1) Based on the above optimization conditions, 600 μL of CMA signal probe solution (with a CMA signal probe concentration of 2 mg / mL) was the optimal experimental condition. 600 μL of CMA signal probe solution (with a CMA signal probe concentration of 2 mg / mL, PBS as solvent, pH=7.4) and 100 μL of FA capture probe solution (with a FA capture probe concentration of 2 mg / mL, PBS as solvent, pH=7.4) were added simultaneously to 1 mL of solution containing 1×10⁻⁶ A549 cells. 4The cell solution was prepared at / mL, and the incubation volume was increased to 2 mL with PBS (pH=7.4). The mixture was then incubated at 37℃ for 1 h to obtain CMA-A549-FA.

[0075] (2) CMA-A549-FA was separated by adsorption with a strong magnet and washed twice with PBS (pH=7.4).

[0076] (3) The enriched reactants were dispersed in 255 μL of PBS solution containing H2O2 (35 μL, 20 mM) and TMB (20 μL, 11 mM) and reacted at room temperature in the dark for 1 h to obtain CMA-A549-FA solution. The pH of the PBS was 4, 5, 6, 7, 8, 9 and 10.

[0077] (4) 100 μL of CMA-A549-FA solution was dropped onto the screen-printed electrode. The obtained electrical signal was detected using differential pulse voltammetry (DPV) in the range of 0.2~0.4 V, with a pulse amplitude of 50 mV and a pulse width of 50 ms. Substituting these values ​​into the linear relationship formula between the DPV signal and the number of pulses (y=0.9228x+9.3364, R...), the signal was analyzed. 2 =0.9971), obtaining the pH values ​​for the reactions of CMA-A549-FA with TMB and H2O2, and the DPV signal intensity under this method. Results are as follows: Figure 10 As shown, pH 8 is optimal.

[0078] 3. The specific method for optimizing the incubation time between cells and aptamers is as follows: (1) Based on the above optimization conditions, the optimal experimental conditions were 600 μL of CMA signal probe solution (CMA signal probe concentration in the solution was 2 mg / mL), and pH=8 when CMA-A549-FA reacted with TMB and H2O2. 600 μL of CMA signal probe solution (CMA signal probe concentration in the solution was 2 mg / mL, solvent was PBS, pH=7.4) and 100 μL of FA capture probe solution (FA capture probe concentration in the solution was 2 mg / mL, solvent was PBS, pH=7.4) were added simultaneously with 1 mL of solution containing 1×10⁻⁶ A549 cells. 4 In a 1 / mL cell culture solution, the final incubation volume was increased to 2 mL with PBS (pH=7.4), and the cells were incubated at 37℃ for 20, 30, 40, 50, 60, and 70 min respectively to obtain CMA-A549-FA.

[0079] (2) CMA-A549-FA was separated by adsorption with a strong magnet and washed twice with PBS (pH=7.4).

[0080] (3) The enriched reactants were dispersed in 255 μL of PBS solution (pH=8) containing H2O2 (35 μL, 20 mM) and TMB (20 μL, 11 mM) and reacted at room temperature in the dark for 1 h to obtain CMA-A549-FA solution.

[0081] (4) 100 μL of CMA-A549-FA solution was dropped onto the screen-printed electrode. The obtained electrical signal was detected using differential pulse voltammetry (DPV) in the range of 0.2~0.4 V, with a pulse amplitude of 50 mV and a pulse width of 50 ms. Substituting these values ​​into the linear relationship formula between the DPV signal and the number of pulses (y=0.9228x+9.3364, R...), the signal was analyzed. 2 =0.9971), the DPV signal intensity of cells and aptamers at different incubation times under this method was obtained. The results are as follows: Figure 10 As shown, incubation for 30 minutes is optimal.

[0082] Experimental Example 2: Specificity Experiment of the Method for Detecting CTCs in this Invention Eight clinical samples were selected, including three healthy subjects and five lung cancer patients. CTCs were detected using the method described in Example 3 (with some parameters modified as follows: 600 μL CMA signal probe solution with a concentration of 2 mg / mL; 100 μL FA capture probe solution with a concentration of 2 mg / mL; PBS solution containing H2O2 and TMB at pH 8; and incubation of human blood with CMA signal probe and FA capture probe for 30 min).

[0083] The results are as follows Figure 11 As shown in Figure a. The results indicate a significant difference in DPV curves between healthy subjects and lung cancer patients. The DPV signal in healthy subjects was significantly lower than that in lung cancer patients, and the signal in the healthy group did not differ from the background value. ROC curve analysis ( Figure 11 b) shows that the AUC value of this method in distinguishing lung cancer patients from healthy subjects is 0.8667, indicating that the method has good discriminative ability.

[0084] Experimental Example 3: Specificity Analysis of Cell Detection by CMA-FA Electrochemical Cell Sensor Following the method described in Example 3, the volume of each cell used was 1 mL (1 × 10⁻⁶). 4 / mL), and the cells in each group were tested separately (some parameters were changed to: 600 μL CMA signal probe solution, the concentration of CMA signal probe in the solution was 2 mg / mL; 100 μL FA capture probe solution, the concentration of FA capture probe in the solution was 2 mg / mL; PBS solution containing H2O2 and TMB was pH 8; human blood, CMA signal probe and FA capture probe were incubated for 30 min).

[0085] This system recognizes CTCs through a dual-target EpCAM-MUC1 approach. Experiments showed that A549 cells with high expression of both antigens had significantly higher signal intensity than CACO-2 and SUN719 cells with single antigen expression. Figure 12 a), while low-expressing cells showed the lowest signal. Capture efficiency analysis ( Figure 12 (b) indicates that the number of residual cells in the dual-aptamer probe is significantly lower than that in the single-aptamer system, confirming that the synergistic effect of dual-aptamers can improve the capture efficiency.

[0086] In summary, this invention provides a method for detecting circulating tumor cells (CTCs) using a dual-specificity electrochemical sensor system. The detection system comprises an EpCAM / MUC1 aptamer-coupled dual-specificity magnetic bead CTC capture probe and an EpCAM / MUC1 aptamer-coupled dual-specificity nanozyme CTC signal probe. This detection method utilizes the synergistic effect of the dual aptamers to enhance capture efficiency. This method enables rapid detection of circulating tumor cells in peripheral blood with small sample sizes. Furthermore, this method offers high accuracy and sensitivity, providing accurate results for disease diagnosis.

Claims

1. A signal probe CMA, characterized in that: It is a nanoparticle obtained by grafting MUC1 aptamer and EpCAM aptamer onto the surface of CAT@Fe(SS)MOF; The CAT@Fe(SS)MOF is prepared from ferric chloride hexahydrate, dithiodiacetic acid, catalase, polyvinylpyrrolidone and water. The nucleotide sequence of the MUC1 aptamer is shown in SEQ ID NO.1; the nucleotide sequence of the EpCAM aptamer is shown in SEQ ID NO.

2.

2. The signal probe CMA according to claim 1, characterized in that: The preparation method of the CAT@Fe(SS)MOF includes the following steps: The ferric chloride hexahydrate aqueous solution was poured into the dithiodiacetic acid aqueous solution, and then catalase aqueous solution and polyvinylpyrrolidone aqueous solution were added in sequence to react and obtain CAT@Fe(SS)MOF. The volume ratio of the ferric chloride hexahydrate aqueous solution, dithiodiacetic acid aqueous solution, catalase aqueous solution, and polyvinylpyrrolidone aqueous solution is 1:(1~5):(0.1~0.5):(1~5); And / or, the concentration of the ferric chloride hexahydrate aqueous solution is 5~10 mg / mL; And / or, the concentration of the aqueous solution of dithiodiacetic acid is 5~10 mg / mL; And / or, the concentration of the catalase aqueous solution is 1~5 mg / mL; And / or, the concentration of the polyvinylpyrrolidone aqueous solution is 10~15 mg / mL; And / or, the reaction temperature is 20~40℃, and the reaction time is 10~30min.

3. The method for preparing the signal probe CMA according to claim 1 or 2, characterized in that: It includes the following steps: (a) Add the CAT@Fe(SS)MOF dispersion to the carboxyl activation solution and mix thoroughly to obtain a mixture; (b) Add the MUC1 aptamer solution to the mixture and incubate; (c) After incubation in step (b), add EpCAM aptamer solution and incubate; (d) After incubation, the product is centrifuged, washed, and dried to obtain the final product.

4. The preparation method according to claim 3, characterized in that: In step (a), the carboxyl activation solution is a PBS solution containing EDC and NHS; And / or, in step (a), the volume ratio of the CAT@Fe(SS)MOF dispersion to the carboxyl activation solution is 1:(1~5). And / or, in step (a), the concentration of the CAT@Fe(SS)MOF dispersion is 1~5 mg / mL; And / or, in step (b), the volume ratio of the mixture to the MUC1 aptamer solution is (1~3):1; And / or, in step (b), the concentration of the MUC1 aptamer solution is 100~300 μM; And / or, in step (b), the incubation temperature is 35~37 ℃ and the incubation time is 20~30 h; And / or, in step (c), the volume ratio of the EpCAM aptamer solution to the mixture in step (b) is 1:(1~3); And / or, in step (c), the concentration of the EpCAM aptamer solution is 100~300 μM; And / or, in step (c), the incubation temperature is 35~37 ℃ and the incubation time is 20~30 h.

5. A capture probe FA, characterized in that: It is a nanoparticle obtained by grafting MUC1 aptamer and EpCAM aptamer onto the surface of Fe3O4 nanoparticles with amino groups. The nucleotide sequence of the MUC1 aptamer is shown in SEQ ID NO.1; the nucleotide sequence of the EpCAM aptamer is shown in SEQ ID NO.2; The preparation method of the Fe3O4 nanoparticles with amino groups on the surface includes the following steps: dissolving ferric chloride hexahydrate and anhydrous sodium acetate in ethylene glycol, and then adding hexamethylenediamine to obtain a mixed solution; reacting the mixed solution to obtain Fe3O4 nanoparticles with amino groups on the surface.

6. The capture probe FA according to claim 5, characterized in that: The mass-to-volume ratio of ferric chloride hexahydrate, anhydrous sodium acetate, ethylene glycol and hexamethylenediamine is 1g:(4~5)g:(30~50)mL:(1~5)mL; And / or, the reaction temperature is 200-300℃, and the reaction time is 5-10h.

7. The method for preparing the capture probe FA according to claim 5 or 6, characterized in that: It includes the following steps: (A) Add the MUC1 aptamer solution and the EpCAM aptamer solution to the carboxyl activation solution to activate the carboxyl groups of the aptamers; (B) The MUC1 aptamer solution with activated carboxyl groups was added to the Fe3O4 nanoparticle dispersion and incubated. (C) After incubation in step (B), add EpCAM aptamer solution and incubate; (D) After incubation, the product is centrifuged, washed, and dried to obtain the final product.

8. The preparation method according to claim 7, characterized in that: In step (A), the carboxyl activation solution is a PBS solution containing EDC and NHS; And / or, in step (A), the volume ratio of the MUC1 aptamer solution to the carboxyl activating solution is 1:(1~3). And / or, in step (A), the concentration of the MUC1 aptamer solution is 100~300 μM; And / or, in step (A), the volume ratio of the EpCAM aptamer solution to the carboxyl activating solution is 1:(1~3). And / or, in step (A), the concentration of the EpCAM aptamer solution is 100~300 μM; And / or, in step (B), the volume ratio of the MUC1 aptamer solution to the Fe3O4 nanoparticle dispersion is 1:(1~3). And / or, in step (B), the concentration of the Fe3O4 nanoparticle dispersion is 10~15 mg / mL; And / or, in step (B), the incubation temperature is 35~37 ℃ and the incubation time is 20~30 h; And / or, in step (C), the volume ratio of the EpCAM aptamer solution to the Fe3O4 nanoparticle dispersion is 1:(1~3). And / or, in step (C), the incubation temperature is 35~37 ℃ and the incubation time is 20~30 h.

9. A method for detecting CTCs based on a dual-specificity electrochemical sensor system, characterized in that: It includes the following steps: (1) The solution of the signal probe CMA according to claim 1 or 2 and the solution of the capture probe FA according to claim 5 or 6 are co-incubated with human blood samples to obtain CMA-CTC-FA; (2) Using magnets to enrich CMA-CTC-FA; (3) The enriched CMA-CTC-FA was added to a solution containing H2O2 and TMB to react and obtain a CMA-CTC-FA solution; (4) The electrical signal of the CMA-CTC-FA solution was detected by differential pulse voltammetry, and the number of CTCs was calculated based on the standard curve.

10. The method according to claim 9, characterized in that: In step (1), the concentration of signal probe CMA in the solution is 0.2~2 mg / mL, and the concentration of capture probe FA in the solution is 0.5~2 mg / mL; And / or, in step (1), the solvent in the solution of the signal probe CMA and the solution of the capture probe FA is PBS; And / or, in step (1), the volume ratio of the solution of the signal probe CMA, the solution of the capture probe FA, and human blood is (0.5~0.6):(0.1~0.3):1; And / or, in step (1), the incubation temperature is 35~37℃ and the incubation time is 20~70min; And / or, in step (3), the concentration of H2O2 in the solution is 20~30mM and the concentration of TMB is 10~20mM; And / or, in step (3), the solvent of the solution is PBS, and the pH value of PBS is 4~10; And / or, in step (3), the reaction temperature is 20~40℃ and the reaction time is 1~5h; And / or, in step (4), the voltage detected by the differential pulse voltammetry is 0.2~0.4V, the pulse amplitude is 50~100mV, and the pulse width is 50~100 ms; And / or, in step (4), the standard curve is y = 0.9928x + 9.3364, R 2 =0.9971; x is the number of cells, and y is the DPV signal value.

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