Nanomotor for capturing and detecting CTC with low detection limit, preparation method and application thereof

Abstract

The present invention provides a nanomotor with a low detection limit for capturing and detecting CTCs, its preparation method and application. The preparation method of the nanomotor comprises the following steps: (1) coating a hybrid cell membrane on a nanoparticle, wherein the hybrid cell membrane is composed of a cancer cell membrane and a white blood cell membrane, and the weight ratio of the membrane proteins of the cancer cell membrane and the white blood cell membrane is 2:1; (2) immobilizing urease on one side of the product obtained in step (1); (3) adding an aptamer and an incomplete complementary strand of the aptamer to the product obtained in step (2) for reaction to obtain the nanomotor.

Description

Technical Field

[0001] The present invention belongs to the field of nanomotors, and relates to the technology of using nanomotors for the capture and detection of circulating tumor cells. Specifically, it relates to a nanomotor with a low detection limit for capturing and detecting CTCs, its preparation method and application. Background Art

[0002] The detection of circulating tumor cells (CTCs) has been recognized as a highly promising liquid biopsy technology. However, at present, it has not been widely promoted as an early tumor diagnosis indicator. The reasons for this situation are, on the one hand, that the content of CTCs in blood is extremely low. Many studies have shown that there are only 1 - 10 CTCs per milliliter of blood on average [1] . On the other hand, it is very difficult to exclude the non-specific interference of blood cells in CTC detection, which is another obstacle limiting the accurate capture and detection of CTCs.

[0003] In terms of the capture of CTCs, in previous research work (CN114366811A), the inventor creatively prepared a nanomotor with both self-driven motion characteristics and bionic features. The capture efficiency of CTCs reached 89.3% - 99.7%, solving the problems of the capture efficiency of using nanomotors in CTC capture and the stability of self-driven motion. However, this research has not overcome the problem of the too high detection limit of the current CTC detection technology, and it is impossible to realize the practical application of related technologies for the extremely low content of CTCs in blood.

[0004] Currently, the method widely used for tumor cell detection is immunocytochemical identification. This method is not only cumbersome to operate but also requires cell fixation and permeabilization, which may damage cell viability and function. In addition, in the detection technology of trace or ultra-trace tumor cells, the fluorescent groups used for biological detection are generally organic molecules. They usually have a rigid plane with a large π-conjugated system, and fluorescence quenching occurs when they are in a high concentration or aggregated state, affecting the sensitivity of the probe.

[0005] As far as the inventor knows, the detection limits of current tumor cell detection technologies are usually at the level of 10 - 1000 cells / mL -1 . For example, the technology developed by X. Zhu et al. [2] can achieve a detection limit level of 100 cells / mL -1 . The technology developed by D. Fan et al. [3] can achieve a detection limit level of 15 cells / mL -1 . The technology developed by K. Xiao et al. [4] can achieve a detection limit level of 25 cells / mL -1 . The technology developed by T. Yu et al. [5]The developed technology can achieve a detection limit level of 10 cells / mL -1 That is to say, the current detection technology cannot or can only barely be practically applied to the situation where the content of CTC in blood is only 1 - 10 per milliliter.

[0006] To address the above deficiencies, there is an urgent need in this field for a CTC detection technology with a detection limit as low as 1 cell / mL -1 for the situation where the content of CTC in blood is only 1 cell / mL -1 .

[0007] References:

[0008] [1] S.B. Cheng, M. Wang, C. Zhang, M.M. Chen, W.H. Huang, Flexible three - dimensional net for intravascular fishing of circulating tumor cells, Anal. Chem., 92(2020)5447 - 5455.

[0009] [2] X. Zhu, J. Yang, M. Liu, Y. Wu, Z. Shen, G. Li, Sensitive detection of human breast cancer cells based on aptamer - cell - aptamer sandwich architecture, Anal Chim Acta, 764(2013)59 - 63.

[0010] [3] D. Fan, C. Wu, K. Wang, X. Gu, Y. Liu, E. Wang, A polydopamine nanosphere based highly sensitive and selective aptamer cytosensor with enzyme amplification, Chem Commun (Camb), 52(2016)406 - 409.

[0011] [4] K. Xiao, J. Liu, H. Chen, S. Zhang, J. Kong, A label-free and high-efficient GO-based aptasensor for cancer cells based on cyclic enzymatic signal amplification, Biosens Bioelectron, 91(2017)76-81.

[0012] [5] T. Yu, P. P. Dai, J. J. Xu, H. Y. Chen, Highly Sensitive Colorimetric Cancer Cell Detection Based on Dual Signal Amplification, ACS Appl Mater Interfaces, 8(2016)4434-4441. Summary of the Invention

[0013] In view of the requirements of the prior art, the object of the present invention is to provide a detection technology for CTC with a detection limit as low as 1 cell / mL, considering that the content of CTC in blood is only 1 cell / mL. -1 In this case, the detection limit is as low as 1 cell / mL. -1 for CTC.

[0014] To achieve the above object, the technical solution provided by the present invention is as follows:

[0015] A preparation method of a nanomotor with a low detection limit for capturing and detecting CTC, the method comprising the following steps:

[0016] (1) Coating a hybrid cell membrane on nanoparticles, the hybrid cell membrane being composed of a cancer cell membrane and a white blood cell membrane, and the weight ratio of the membrane proteins of the cancer cell membrane and the white blood cell membrane being 2:1;

[0017] (2) Fixing urease on one side of the product obtained in step (1);

[0018] (3) Adding an aptamer and an incomplete complementary strand of the aptamer to the product obtained in step (2) for reaction to obtain a nanomotor;

[0019] After the nanomotor captures circulating tumor cells, the circulating tumor cells are detected with a probe;

[0020] The sequence of the aptamer is cholesterol-modified at the 3' end of the sequence shown in SEQ ID NO: 1, specifically: 5′-ACA GCATCC CCATGT GAA CAATCG CAT TGT GAT TGT TAC GGT TTC CGC CTC ATGGAC GTG CTG-cholesterol-3′;

[0021] The incomplete complementary strand of the aptamer is as shown in SEQ ID NO: 2, specifically: 3′-TGT CGTAGGGGTACATTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GAG TAC CTG CAC GAC-5′;

[0022] The preparation method of the probe is as follows:

[0023] Add the complementary strand with an alkynyl group at the 5' end and Compound I into water and dimethyl sulfoxide respectively. Mix the obtained aqueous solution and dimethyl sulfoxide solution in equal volumes at the same solute concentration, and then add CuSO4 and sodium ascorbate in a molar ratio of 1:2 for reaction to obtain the probe;

[0024] The sequence of the complementary strand with an alkynyl group at the 5' end is alkynyl-modified at the 5' end of the sequence shown in SEQ ID NO: 3, specifically: 5′-alkynyl-ACA GCATCC CCATGT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA CTC ATGGAC GTG CTG-3′;

[0025] The Compound I is as shown in Formula <1>;

[0026]

[0027] In the process of research of the present invention, it is found that even in the previous research results of the inventors, namely CN 114366811A, the hybrid membrane composed of three cell membranes has a higher capture rate in CN 114366811A; however, in the present invention, when the aptamer of the present invention is added and the hybrid membrane composed of three cell membranes is selected to prepare the nanomotor of the present invention, the capture efficiency for CTC decreases significantly. The possible reason is that when the hybrid membrane composed of three cell membranes binds to the aptamer and the incomplete complementary strand, the binding is unbalanced or unstable, resulting in a decrease in the self-driven motion stability. Fortunately, in the present invention, when only the cancer cell membrane and the white blood cell membrane are used to form the hybrid cell membrane, the capture efficiency for CTC is increased instead, reaching 96.7%. The possible reason is that the addition of the aptamer and the incomplete complementary strand increases the binding stability of the substances on the surface of the nanomotor.

[0028] Meanwhile, the inventors also found that the selection of the aptamer has a greater impact on the detection limit of CTC. As shown in an example of the present invention, when the aptamer is replaced, the obtained detection limits can only reach 100 and 125 cells mL -1 levels, making the corresponding technical solutions difficult to be used for the actual detection application of CTC.

[0029] As an implementable technical solution of the present invention, in step (1), the nanoparticles include Fe3O4 magnetic nanoparticles.

[0030] As an implementable technical solution of the present invention, the hybrid cell membrane is obtained by extruding and fusing different cell membranes through a polycarbonate porous membrane.

[0031] As an implementable technical solution of the present invention, when immobilizing urease, first uniformly adsorb the product obtained in step (1) on a polylysine-modified cell culture plate, then add N-hydroxysulfosuccinimide biotin and streptavidin to the culture plate for reaction, and then add biotin-modified urease for reaction; the biotin-modified urease is obtained by dissolving urease in PBS buffer and adding N-hydroxysulfosuccinimide biotin for reaction.

[0032] As a preferred technical solution of the present invention, the addition amounts of the aptamer and the incomplete complementary strand of the aptamer are the same; the aptamer and the incomplete complementary strand of the aptamer are in excess with respect to the binding force of the product obtained in step (2) for the two.

[0033] As a preferred technical solution of the present invention, when preparing the probe, after the reaction is completed, unreacted compound I is removed by dichloromethane extraction, and then purified by reverse HPLC.

[0034] As a preferred technical solution of the present invention, when preparing the probe, the reaction time is 24 h and the reaction temperature is 20-30 °C.

[0035] Another object of the present invention is to provide a nanomotor with a low detection limit for capturing and detecting CTCs prepared by the above preparation method.

[0036] Another object of the present invention is to provide the application of the above nanomotor in capturing and detecting CTCs. Specifically, the application method is as follows: adding the bionic nanomotor and urea to a sample, using the nanomotor to bind to circulating tumor cells, separating the captured circulating tumor cells by magnetic field, then adding a probe to the remaining reaction and establishing a standard detection equation based on the corresponding relationship between the fluorescence intensity of the probe and the number of circulating tumor cells, and using the standard detection equation to detect and quantify the concentration of circulating tumor cells.

[0037] Advantages of the present invention:

[0038] The nanomotor obtained by the present invention can be used for the capture and detection of CTCs. The capture efficiency for CTCs can reach 96.7%, and the detection limit is as low as 1 cells mL -1 , having good practical application prospects. Description of the Drawings

[0039] Figure 1 is a schematic diagram of the preparation process of the nanomotor of the present invention;

[0040] Figure 2 is a schematic diagram of the process of detecting CTCs of the present invention;

[0041] Figure 3 is a schematic diagram of the preparation process of the probe of the present invention;

[0042] Figure 4 is the movement trajectory of the nanomotor in urea solutions with different concentrations;

[0043] Figure 5 is the mean square displacement of the nanomotor in urea solutions with different concentrations;

[0044] Figure 6 is the movement speed of the nanomotor in urea solutions with different concentrations. Detailed Embodiments

[0045] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and should not be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the protection scope of the present invention.

[0046] In the following examples of the present invention, unless otherwise specified, other abbreviations and their meanings are as follows, except that the abbreviations are recorded in parentheses after the relevant substances:

[0047] CTC: Circulating tumor cells

[0048] Sulfo-NHS-biotin: N-Hydroxysulfosuccinimide biotin

[0049] Fe3O4@HM: Fe3O4 nanoparticles coated with hybrid cell membranes

[0050] Fe3O4@HM / Ure: Fe3O4@HM immobilized with urease

[0051] Fe3O4@HM / Ure-Ap: Fe3O4@HM / Ure modified with aptamer

[0052] Example 1

[0053] 1. Extraction of cell membranes

[0054] Prepare Hepes B (2.38 g / L Hepes, 0.476 g / L MgCl2, 0.292 g / L EDTA, 0.154 g / L DTT, 0.746 g / L KCl, pH 7.6) and Hepes C (11.914 g / L Hepes, 5.844 g / L NaCl, 13.492 g / L KCl, pH 7.6) buffers. Culture HepG2 cells in a petri dish, centrifuge to collect cancer cells after digestion, suspend them in a mixture of Hepes B buffer and 1% protease inhibitor, homogenize the cells several times after cooling in an ice bath for 5 minutes, and remove the cell nuclei. Repeat the above steps, and finally collect 8 - 10 mL of supernatant. Prepare sucrose solutions with concentrations of 30%, 40%, and 55% (w / v) using Hepes B buffer, add them to centrifuge tubes in order of decreasing concentration, and slowly add the collected supernatant to the sucrose density gradient column. Centrifuge at high speed at 4°C, collect the required sample band, resuspend it repeatedly with Hepes C buffer, and centrifuge. Finally, resuspend the obtained cancer cell membranes in Hepes C buffer for storage. Collect whole blood from healthy volunteers, store it in a heparin sodium anticoagulant blood collection tube, collect white blood cells using gradient centrifugation, and then break the cells and collect white blood cell membranes using the above method.

[0055] 2. Preparation of hybrid cell membranes

[0056] Add cancer cell membranes to white blood cell membranes according to a membrane protein weight ratio of 2:1. Slowly stir each sample at room temperature for several minutes, then perform ultrasonic treatment for 3 minutes, and finally extrude and fuse through 400 nm and 200 nm polycarbonate porous membranes, and centrifuge to collect cancer cell - white blood cell hybrid membranes.

[0057] 3. Preparation of Hybrid Membrane-Coated Nanocarriers

[0058] Take Fe3O4 magnetic nanoparticles and add them to an excessive amount of cancer cell - white blood cell hybrid membranes. Dilute the mixture and perform ultrasonic treatment in an ice bath for 3 minutes, and then pass it through polycarbonate porous membranes with pore sizes of 400 nm and 200 nm. Collect the hybrid membrane-coated biomimetic nanocarriers (Fe3O4@HM) by high-speed centrifugation, wash them three times with buffer solution, and remove the excess cell membranes.

[0059] 4. Immobilized Enzyme

[0060] Dissolve urease in PBS buffer solution (1 mg / mL), and then add an equal volume of Sulfo-NHS-biotin (16 μM) to the enzyme solution and react at room temperature for half an hour. Filter to remove the unreacted Sulfo-NHS-biotin, and centrifuge to obtain biotinylated urease (Urease-biotin) dissolved in PBS buffer solution and store it at 4 °C for standby. Add an appropriate amount of Fe3O4@HM biomimetic nanocarrier suspension to a 12-well cell culture plate modified with polylysine, and make the biomimetic nanocarriers evenly adsorb on the bottom of the culture plate by centrifugation. After reacting at room temperature for one hour, remove the supernatant and wash it several times with PBS to remove the unadsorbed biomimetic nanocarriers. Subsequently, add Sulfo-NHS-biotin (160 μM) and streptavidin (160 μM) to the culture plate successively and react at room temperature for 1 hour, and wash each step of the reaction with PBS. Finally, add Urease-biotin and react at room temperature for 1 hour. Immobilize urease on the unblocked side of the biomimetic nanocarriers through the biocompatible interaction between biotin and streptavidin, and slowly blow and wash it with a pipette to release it from the culture plate to obtain the biomimetic nanomotor Fe3O4@HM / Ure.

[0061] 5. Aptamer Modification

[0062] The nucleic acid aptamer 5′-ACA GCA TCC CCA TGT GAA CAA TCG CAT TGT GAT TGT TACGGT TTC CGC CTC ATG GAC GTG CTG-cholesterol-3′ was selected as the related affinity molecule (aptamer), and 3′-TGT CGTAGG GGT ACA TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GAG TAC CTGCAC GAC-5′ was used as the incomplete complementary strand (anti-aptamer) of the aptamer. Equal amounts of aptamer and anti-aptamer were added to the Fe3O4@HM / Ure biomimetic nanomotor suspension and reacted at room temperature for half an hour. The hydrophobic interaction of cholesterol at the 3′ end of the aptamer was used to fix the stem-loop structure formed by the aptamer and anti-aptamer on the surface of the nanomotor. The unfixed aptamer and anti-aptamer were washed with buffer to obtain Fe3O4@HM / Ure-Ap, that is, the nanomotor.

[0063] 6. Preparation of Fluorescent Probe

[0064] The structure of TPEN3 is as Figure 3 shown. Referring to the literature method (Ma et al. ACSSens. 2018, 3, 320–326), TPE derivative 1 was prepared, and then sodium azide (NaN3) was used to replace the Br group to prepare TPE derivative TPEN3 with an N3 group. The anti-aptamer complementary strand with an alkyne group at the 5′ end (5′-alkynyl-ACA GCATCC CCATGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA CTCATG GAC GTG CTG-3′) and TPEN3 were respectively added to aqueous solution and DMSO. After mixing the obtained aqueous solution (0.5 μM, 500 μL) and dimethyl sulfoxide solution (0.5 μM, 500 μL), CuSO4 (0.3 nmol) and sodium ascorbate (0.6 nmol) were added and reacted at room temperature (20 - 30 °C) for 24 hours. After the reaction was completed, the unreacted TPEN3 was removed by dichloromethane extraction, and the product was purified by reverse HPLC to obtain a fluorescent probe (Probe) with TPEN3 at the 5′ end.

[0065] Comparative Example 1

[0066] When preparing the nanomotor, except that the hybrid cell membrane was changed to be composed of cancer cell membrane, white blood cell membrane and red blood cell membrane according to the membrane protein weight ratio of 4:2:1, the rest was the same as in Example 1.

[0067] Comparative Example 2

[0068] When modifying the aptamer, except that the aptamer was replaced with 5′-TGA GGT AGT AGG TTG TGT GGTT GCAGTT GAT CCT TTG GAT ACC CTG G-cholesterol-3′, and other sequences were adaptively adjusted based on Reference Example 1, the rest was the same as in Example 1.

[0069] Comparative Example 3

[0070] When modifying the aptamer, except that the aptamer was replaced with 5′-ATCTAACTGC TGCGCCGCCG GGAAAATACTGTACGGTTAGATTTTTTTTTTTTT ACCTCAGCAGTTAGGCCATTTT-cholesterol-3′, and other sequences were adaptively adjusted based on Reference Example 1, the rest was the same as in Example 1.

[0071] Experimental Example 1

[0072] (1) Characterization of the hybrid membrane

[0073] To characterize the formation of the hybrid membrane, DSPE-PEG-FITC dye and leukocyte membrane were mixed and reacted for several hours, and DSPE-PEG-Cy5 dye and cancer cell membrane were mixed and reacted for several hours. After the fluorescence labeling was completed, the excess dye was removed by centrifugation. The fluorescently labeled cell membranes were fused to form hybrid cell membranes in the same way and dropped onto glass slides. FITC was excited at 488 nm by CLSM, and the green fluorescence at 525 nm was collected. Cy5 was excited by a 649 nm laser, and the red fluorescence at 670 nm was collected. The unfused cell membranes were used as a control, and the formation of the two hybrid membranes was determined by the superposition of the fluorescence colors. The results showed that the hybrid membranes were successfully formed.

[0074] (2) Coating characterization and stability evaluation of the hybrid cell membrane

[0075] The Fe3O4@HM was counterstained with phosphotungstic acid, and its morphology was characterized by TEM to observe the coating of the hybrid cell membrane on the nanocarrier. To verify the core-shell structure of the biomimetic nanocarrier, the hybrid cell membrane was labeled with FITC, and the fluorescence distribution of FITC was observed by CLSM. The hydrodynamic diameter and Zeta potential of the biomimetic nanocarrier were measured by DLS to compare the changes before and after the coating of the hybrid cell membrane. The effects of the hybrid cell membrane coating process on the drug loading rate and encapsulation efficiency were tested. The nanocarriers coated with single cell membrane and hybrid cell membrane were mixed with the loading buffer and heated at 100 °C for 10 minutes, and the samples containing equal amounts of protein were loaded onto a 10% SDS-PAGE gel and electrophoresed at 120 KV. After electrophoresis, the gel was stained with Coomassie Brilliant Blue solution and rinsed, and decolorized overnight with glacial acetic acid to analyze the cell membrane proteins of the biomimetic nanocarrier. The proteins were transferred to a nitrocellulose membrane, blocked at room temperature for several hours, and developed by enhanced chemiluminescence detection. The experiment was repeated 3 times, and the resulting films were semi-quantitatively analyzed for protein bands using a multi-functional pathological image analysis system to measure the optical density values. The biomimetic nanocarriers were dispersed in PBS buffer and bovine serum solution and stored for two weeks, and the particle size changes were measured by DLS to verify their long-term stability.

[0076] (3) Characterization of immobilized enzyme

[0077] To verify the unilateral distribution of urease on the biomimetic nanocarrier, colloidal gold with a particle size of 5 nm was mixed with the enzyme solution, and the enzyme was labeled by adjusting the pH. The enzyme labeled with colloidal gold was immobilized on one side of the biomimetic nanocarrier by the same method, and the enzyme distribution was observed by TEM after counterstaining with phosphotungstic acid. The amount of immobilized enzyme was calculated using a BCA protein quantification test kit; the enzyme activity was calculated using the corresponding kit, and the optimal pH and temperature were determined.

[0078] (4) Characterization of the motion characteristics of nanomotors

[0079] The prepared nanomotors were separately dispersed in 100 μL of PBS buffer containing different concentrations of urea. Subsequently, the suspension was added to a culture dish, observed with an optical microscope, and the motion of the nanomotors was recorded with a CCD camera for 10 seconds, with a relative frame rate of 25 fps. The TrackMate software was used to track the motion trajectories of the nanomotors, and then the Matlab software was used to extract and output their trajectories, and further calculate the motion characteristic parameters such as the motion speed, mean square displacement, and diffusion coefficient of the nanomotors. To ensure relatively constant enzyme activity, all recorded videos were of the motion of the biomimetic nanomotors within 3 minutes after the addition of urea. The results are shown in Figures 4-6 as follows.

[0080] The nanomotors were dispersed in PBS buffer and bovine serum solution and stored for two weeks. The long-term storage stability was verified by measuring the particle size change through DLS. Meanwhile, to investigate the binding stability, the nanomotors were diluted and dispersed in PBS buffer and placed on a shaker (2000 rpm / min) for a period of time, and the maintenance effect of the self-propelled motion function of the obtained nanomotors was recorded. The long-term storage and the stability of the self-propelled motion function of Example 1 and Comparative Example 1 were evaluated. The long-term storage was investigated with a 10% change in particle size as the index; the stability of the self-propelled motion function was investigated by taking the ratio of the number of biomimetic nanomotors still having the self-propelled motion ability to the total number when the nanomotors were shaken on the shaker for 30 minutes and observed in the microfluidic device. The results are shown in Table 1.

[0081] Table 1

[0082]

[0083] (5) CTC capture

[0084] Referring to the method in the literature (DOI: 10.1021 / acsnano.7b08355), the CTC active capture efficiency experiments of Example 1 and Comparative Example 1 were carried out, and the results are shown in Table 2:

[0085] Table 2

[0086]

[0087] (6) CTC detection

[0088] Whole blood from healthy volunteers and cancer patients was collected. A certain number of HepG2 cells were added to the whole blood of healthy people to simulate the blood of cancer patients. The nanomotors were used to carry out CTC capture and detection experiments on the simulated and real cancer patient blood. A certain number of CTCs were added to the blood samples to prepare a series of standard samples (20 cells / mL, 50 cells / mL, 100 cells / mL, and 200 cells / mL). The biomimetic nanomotors (100 μg / mL) and urea (5 mM) were added to 400 μL of the standard samples. The biomimetic nanomotors were used to bind to the circulating tumor cells, and the captured circulating tumor cells were separated by magnetic field. Probes (20 μM) were added to the remaining reaction, and a standard detection equation was established through the corresponding relationship between the fluorescence intensity of the probes and the number of circulating tumor cells. The same detection steps were used for the unknown concentration of the sample to be detected, and the CTC concentration was quantified using the standard detection equation. The captured cells were stained by immunocytochemistry to identify cancer cells and other blood cells, and the capture efficiency of the nanomotors in the blood was calculated. Using the fluorescence signal of the blank blood sample without adding CTC as the noise signal, the CTC concentration was continuously reduced to measure the detection signal, and the detection limit of CTC was determined when the signal-to-noise ratio was 3. The results are shown in Table 3.

[0089] Table 3

[0090]

Claims

1. A preparation method of a nanomotor with a low detection limit for capturing and detecting CTCs, characterized in that, The method includes the following steps: (1) Coating a hybrid cell membrane on nanoparticles, where the hybrid cell membrane is composed of a cancer cell membrane and a white blood cell membrane, and the weight ratio of the membrane proteins of the cancer cell membrane and the white blood cell membrane is 2:1; (2) Fixing urease on one side of the product obtained in step (1); (3) Adding an aptamer and an incomplete complementary strand of the aptamer to the product obtained in step (2) for reaction to obtain a nanomotor; After the nanomotor captures circulating tumor cells, a probe is used to detect the circulating tumor cells; The aptamer is: 5′-ACA GCA TCC CCA TGT GAA CAA TCG CAT TGT GAT TGT TAC GGTTTC CGC CTC ATG GAC GTG CTG-cholesterol-3′; The incomplete complementary strand of the aptamer is: 3′-TGT CGT AGG GGT ACA TTT TTT TTT TTT TTT TTTTTT TTT TTT TTT TTT GAG TAC CTG CAC GAC-5′; The preparation method of the probe is: Adding a 5'-terminal alkynyl complementary strand and Compound I into water and dimethyl sulfoxide respectively, mixing the obtained aqueous solution and dimethyl sulfoxide solution in equal volume at the same solute concentration, and then adding CuSO4 and sodium ascorbate with a molar ratio of 1:2 for reaction to obtain the probe; The 5'-terminal alkynyl complementary strand is: 5′-alkynyl-ACA GCATCC CCATGT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA CTC ATG GAC GTG CTG-3′; The Compound I is as shown in formula <1>; 2. The preparation method according to claim 1, characterized in that, In step (1), the nanoparticles include Fe3O4 magnetic nanoparticles.

3. The preparation method according to claim 1, characterized in that, The hybrid cell membrane is obtained by extruding and fusing different cell membranes through a polycarbonate porous membrane.

4. The preparation method according to claim 1, characterized in that, When fixing urease, first evenly adsorb the product obtained in step (1) on a polylysine-modified cell culture plate, then add N-hydroxysulfosuccinimide biotin and streptavidin to the culture plate for reaction, and then add biotin-modified urease for reaction; the biotin-modified urease is obtained by dissolving urease in PBS buffer and adding N-hydroxysulfosuccinimide biotin for reaction.

5. The preparation method according to claim 1, characterized in that, The addition amounts of the aptamer and the incomplete complementary strand of the aptamer are the same; the aptamer and the incomplete complementary strand of the aptamer are in excess relative to the binding force of the product obtained in step (2) for the two.

6. The preparation method according to claim 1, characterized in that, When preparing the probe, after the reaction is completed, unreacted Compound I is removed by dichloromethane extraction, and then purified by reverse HPLC.

7. The preparation method according to claim 1, characterized in that, When preparing the probe, the reaction time is 24 h, and the reaction temperature is 20 - 30 °C.

8. A nanomotor with a low detection limit for capturing and detecting CTCs prepared by the preparation method according to any one of claims 1-7.

9. Use of the nanomotor according to claim 8 in the preparation of a preparation for capturing and detecting CTCs.

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