A circulating tumor cell detection kit and a preparation method and use method thereof

Abstract

The application discloses a circulating tumor cell detection kit and a preparation method and a use method thereof. A capture probe takes ferroferric oxide nano as a core, and hyaluronic acid is used as a modification material to coat the periphery of the ferroferric oxide nano, so that the capture and magnetic separation of the circulating tumor cells are realized. A detection probe takes CdS@Au nano particles as a core, CuS@Au is generated through the replacement of cadmium ions by copper ions, and folate is used as a modification material to realize the targeting of the CdS@Au to the circulating tumor cells. The application realizes the instant detection of the circulating tumor cells, reduces the detection cost of the circulating tumor cells and the technical requirements of an operator, and can effectively solve the problem of how to use a multi-mode detection method to detect the circulating tumor cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanobiological sensing, in particular to an application of a method for instant detection of circulating tumor cells based on ion exchange technology. BACKGROUND

[0002] Circulating tumor cells are malignant cells that fall off from tumor tissues in peripheral blood or lymphatic system. As an important indicator of cancer cell metastasis, circulating tumor cells can be beneficial to prove the occurrence and metastasis of cancer. Studies have shown that circulating tumor cells can be found in peripheral blood in the early stage of cancer occurrence. Therefore, monitoring circulating tumor cells in blood can provide an important means for early diagnosis of cancer. However, there are two difficult problems in the detection of circulating tumor cells in peripheral blood: one is the challenge of the small number of circulating tumor cells in peripheral blood. According to research reports, there are only a few to several hundred circulating tumor cells in one milliliter of blood, far less than blood cells. In order to solve this problem, it is very important to enrich circulating tumor cells in peripheral blood, and a common method for enriching circulating tumor cells is to use the magnetism of magnetic nanoprobes to realize the enrichment of tumor cells. The other problem is that the complex biological environment in peripheral blood poses a great threat to the detection of circulating tumor cells. Therefore, in order to improve the accuracy of the detection results, we can separate circulating tumor cells from peripheral blood. Similarly, because of the advantages of high capture efficiency, fast response and easy operation, immunomagnetic separation is also a good choice.

[0003] At present, instant detection methods based on different readout signals have been used for the detection of circulating tumor cells, such as colorimetric method, electrochemical method, mass spectrometry and blood glucose meter method, etc. The advantages of these detection methods are accuracy, sensitivity, etc., but there are also many deficiencies such as long detection time, high detection cost, and large detection instruments which are not conducive to on-site detection. Therefore, we urgently need to develop new instant detection methods for the detection of circulating tumor cells.

[0004] Some simple signal-based platforms, such as temperature, pressure, pH, etc., have been used in biosensing platforms, but the photothermal detection platform based on temperature and the dual-mode real-time detection platform based on pressure have not been applied to the detection of circulating tumor cells. In the photothermal detection platform, the photothermal effect of the photothermal reagent plays a key role in the accuracy of the detection platform. Among them, the photothermal reagent mainly has two sources: one is that the photothermal reagent prepared in the external environment is directly applied to the detection platform. The other is to generate the photothermal agent in situ in the introduced detection material. The advantage of this method is to improve the accuracy of the detection result. However, the conditions for generating the photothermal reagent in situ are often harsh, so we need to develop a new mild in-situ generation method, and ion exchange reaction is a good choice. CuS nanomaterials as a good photothermal reagent have attracted widespread attention. We take CdS nanoparticles as the core, realize the in-situ generation of CuS nanoparticles through simple ion exchange, and deposit gold nanoparticles on the surface of CdS nanomaterials to realize the modification of the target head-folic acid of the target circulating tumor cells. Through signal amplification, the accuracy of the detection result can be greatly improved. SUMMARY

[0005] The purpose of the present application is to provide a method for preparing and capturing and detecting circulating tumor cells by real-time detection technology based on ion exchange technology to generate folic acid modified CdS@Au nanoprobe, preparation method and application.

[0006] The present application is realized as follows:

[0007] A circulating tumor cell detection kit comprises FA-CdS@Au nano-detection probe and Fe3O4@HA nano-capture probe.

[0008] Further improvement, the copper ion solution is a copper chloride solution, and the mass concentration of the copper chloride solution is 1 mg / mL-2 mg / mL.

[0009] Further improvement, the mass ratio of the FA-CdS@Au nano-detection probe and the Fe3O4@HA nano-capture probe is 1-4:1.

[0010] A preparation method of a circulating tumor cell detection kit comprises the following steps:

[0011] Step one, preparing FA-CdS@Au nano-detection probe;

[0012] Step two, preparing Fe3O4@HA nano-capture probe.

[0013] Further improvement, the preparation method of the FA-CdS@Au nano-detection probe is as follows:

[0014] Step 1.1, sequentially add silica nanospheres, sodium citrate aqueous solution, cadmium chloride solution, ammonia and thiourea in a container, react at 80℃ for 3 hours to obtain SiO2@CdS nanoparticles;

[0015] Step 1.2, add SiO2@CdS nanoparticles into sodium hydroxide solution, remove silica at 90℃ for 3 hours to obtain CdS nanoparticles;

[0016] Step 1.3, add CdS nanoparticles into deionized water, then sequentially add polyvinylpyrrolidone solution, tetrachloroauric acid solution and ascorbic acid solution, react at room temperature for three hours to obtain CdS@Au nanoparticles;

[0017] Step 1.4, disperse CdS@Au nanoparticles in deionized water, then add FA-PEG-SH and react in dark conditions for 24h to obtain the FA-CdS@Au nano-detection probe.

[0018] Further improvement, the specific steps of step 1.1 are as follows: disperse silica nanospheres in 100 ml deionized water, ultrasonic dispersion is uniform, then transfer to a round-bottom flask, add sodium citrate aqueous solution in the round-bottom flask and stir for 5 min, then add cadmium chloride solution and stir again for 5 min, then slowly add 2 ml ammonia and thiourea and react for 5 min, then transfer the round-bottom flask to an oil bath at 80℃ and react for 3 hours, then process to obtain SiO2@CdS nanoparticles; wherein the mass / volume ratio of silica nanospheres, deionized water, sodium citrate aqueous solution, cadmium chloride solution, ammonia and thiourea is 150:100:1:0.5:2:2 mg / ml; wherein the concentration of sodium citrate aqueous solution, cadmium chloride and thiourea is 1 mol / L; the mass concentration of ammonia is 28%;

[0019] In step 1.2, the concentration of sodium hydroxide solution is 0.5 mol / L;

[0020] In step 1.3, the mass / volume ratio of CdS nanoparticles, deionized water, polyvinylpyrrolidone solution, tetrachloroauric acid solution and ascorbic acid solution is 10:10:1:3:4 mg / ml; wherein the mass concentration of polyvinylpyrrolidone solution and tetrachloroauric acid solution is 0.1%;

[0021] In step 1.4, the mass concentration ratio of CdS@Au nanoparticle solution to FA-PEG-SH solution is 2:1, and the volume ratio is 1:1.

[0022] Further improvement, the preparation method of the Fe3O4@HA nano-capture probe is as follows:

[0023] Step 2.1, add FeCl3.6H2O, FeCl2.4H2O and HA into a container, then add deionized water, stir until uniform, and then heat to reflux to 100 degrees Celsius under nitrogen protection for 15 min to obtain a mixed solution;

[0024] Step 2.2, add a solution of NaOH to the mixed solution, and react at 100 degrees Celsius under nitrogen protection for 1 h to obtain a reaction solution;

[0025] Step 2.3, transfer the reaction solution to a reaction kettle, and age at 150 degrees Celsius for 24 h. After aging, the solution is loaded into a dialysis bag and dialyzed for 2 days to obtain Fe3O4@HA nanocapture probes.

[0026] Further improvement, in step 2.1, the mass-volume ratio of FeCl3.6H2O, FeCl2.4H2O, HA and deionized water is 0.36:0.132:30:32.5 g / ml;

[0027] In step 2.2, the weight ratio of NaOH in the NaOH solution to the mass of HA is 1000:30.

[0028] A method for using the above-mentioned circulating tumor cell detection kit, characterized in that it comprises the following steps:

[0029] S1, add cancer cells to a multi-well plate for plating;

[0030] S2, add FA-CdS@Au nanodetection probes and Fe3O4@HA nanocapture probes, then put them into a shaker for co-incubation for 30 min, then use magnetic separation to wash away the excess sample solution, then add PBS solution to obtain a to-be-detected solution, then add copper chloride solution to the to-be-detected solution, and then ultrasonic reaction for 10 min to obtain a detection solution;

[0031] S3, detect the detection solution:

[0032] S3.1, when the detection method is photothermal method: use a 1064 nm laser to irradiate the detection solution for 10-15 min, use an infrared thermal imager to collect temperature changes and record temperature differences;

[0033] 3.2, when the detection method is pressure method: weigh 20-100 mg of ammonium bicarbonate in a sealed pressure bottle, add the to-be-detected solution, and then use a 1064 nm laser to irradiate for 10-15 min, and a barometer records the pressure changes at different time periods.

[0034] Further improvement, in step S1, the concentration of cancer cells is 10 / mL-10 W / mL; the concentration ratio of FA-CdS@Au nano-detection probe and Fe3O4@HA nano-capture probe is 800 μg / mL:200 μg / mL, the volume ratio is 1:1, the added volume of FA-CdS@Au nano-detection probe and Fe3O4@HA nano-capture probe is 1-4:1; the volume ratio of FA-CdS@Au nano-detection probe and cancer cells is 1:9.

[0035] The present application detects circulating tumor cells by double probe recognition strategy, the capture probe Fe3O4@HA takes Fe3O4 magnetic nanoparticles as the core, and HA as the target head can specifically recognize and combine CD44, and can realize the enrichment and separation of rare circulating tumor cells in blood. The detection probe FA-CdS@Au can specifically recognize and combine FR receptors, and after adding copper chloride solution which can provide copper ions, ion exchange occurs to generate FA-CuS@Au with excellent photo-thermal performance. Through the action of the two kinds of nano probes, the conversion of the biological recognition signal between the receptor ligands to the simple signal (temperature and pressure signal) is realized. The accuracy of the detection result of circulating tumor cells can be effectively improved.

[0036] The present application has the advantages and beneficial effects that: the present application can synthesize the detection probe FA-CdS@Au with uniform shape and good stability, and the capture probe Fe3O4@HA with good magnetic performance. Through the magnetism of the capture probe, the circulating tumor cells are separated from other blood cells in blood; then through the conversion of FA-CdS@Au to FA-CuS@Au, the detection of circulating tumor cells is realized by establishing the relationship between the change of temperature and pressure and the concentration of circulating tumor cells. In this method, the establishment of the double recognition probe strategy can ensure the effective enrichment and separation of circulating tumor cells; and the in-situ generation of FA-CuS@Au with good photo-thermal performance realizes the amplification of the signal, which can improve the accuracy of the experimental result. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 (a) is the synthesis route and principle diagram of FA-CdS@Au in embodiment 1 and Fe3O4@HA in embodiment 2 of the present application;

[0038] Figure 1 (b) is the principle diagram of FA-CdS@Au in embodiment 1 and Fe3O4@HA in embodiment 2 of the present application for double recognition strategy to detect circulating tumor cells;

[0039] Figure 2 is the scanning electron microscope image, transmission electron microscope image, high-resolution transmission electron microscope image and element mapping diagram of CdS@Au NPs in embodiment 2 of the present application;

[0040] Figure 3 is the X-ray diffraction pattern of CdS@Au NPs in Example 2 of the present application;

[0041] Figure 4 is the X-ray photoelectron spectroscopy pattern of CdS@Au NPs in Example 2 of the present application;

[0042] Figure 5 is the UV contrast pattern of CdS@Au NPs and CuS@Au in Example 3 of the present application;

[0043] Figure 6 is the time optimization UV spectrogram of the generation of CuS@Au NPs by the replacement of cadmium ions by copper ions in the copper chloride solution in Example 3 of the present application;

[0044] Figures 7-10 is the various result pattern of the photothermal performance test of the generated CuS@Au NPs under the irradiation of 1064 nm laser in Example 3 of the present application;

[0045] Figures 11-13 is the various result pattern of the pressure performance test of the generated CuS@Au NPs under the irradiation of 1064 nm laser in Example 3 of the present application;

[0046] Figure 14 is the UV spectrogram contrast of FA-CdS@Au with FA and CdS@Au in Example 3 of the present application;

[0047] Figure 15 is the X-ray diffraction pattern of Fe3O4@HA in Example 10 of the present application;

[0048] Figure 16 is the particle size pattern of Fe3O4@HA in Example 10 of the present application;

[0049] Figure 17 is the temperature change pattern of the feasibility of the double recognition strategy detection of circulating tumor cells by using FA-CdS@Au as a detection probe and Fe3O4@HA as a capture probe in Example 10 of the present application;

[0050] Figure 18 is the photothermal result pattern of the detection of circulating tumor cells with different cell concentrations by using FA-CdS@Au as a detection probe and Fe3O4@HA as a capture probe in Example 10 of the present application;

[0051] Figure 19 is the pressure result pattern of the detection of circulating tumor cells with different cell concentrations by using FA-CdS@Au as a detection probe and Fe3O4@HA as a capture probe in Example 10 of the present application. DETAILED DESCRIPTION

[0052] For the purposes of this disclosure, like reference numerals in the description and in the drawings will be understood to refer to like parts, steps and features. In order to facilitate the understanding of this application, a thorough and complete description of the application will be provided below. The preferred embodiments of the application are shown in the attached drawings. However, the application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the specification is for describing particular embodiments only and is not intended to be limiting of the application. Example 1

[0054] Referring to Figure 1 (a) shown, the application provides a preparation method of a folate modified CdS@Au nano-detection probe and a hyaluronic acid modified Fe3O4 nano-capture probe.

[0055] The preparation method of the FA-CdS@Au nano-detection probe comprises the following steps:

[0056] Step one, 150 mg of silica nanoballs is dissolved in 100 ml of deionized water, and after ultrasonic dispersion, the solution is transferred to a round-bottom flask. 1 ml of sodium citrate aqueous solution (1 mol / L) is added to the round-bottom flask and stirred for 5 min, then 0.5 ml of cadmium chloride solution (1 mol / L) is added and stirred for 5 min again, then 2 ml of ammonia and thiourea (1 mol / L) is slowly added and reacted for 5 min, then the round-bottom flask is transferred to an 80°C oil bath for reaction for 3 hours, and after treatment, SiO2@CdS nanoparticles are obtained.

[0057] Step two, the SiO2@CdS nanoparticles obtained by the treatment in step one are uniformly dissolved in 50 ml of sodium hydroxide (0.5 mol / L) aqueous solution, and reacted at 90°C oil bath for 3 hours to remove the silica nanoparticles, and after treatment, CdS nanoparticles are obtained.

[0058] Step three, the CdS nanoparticles (10 mg) prepared in step two are uniformly dispersed in 10 ml of deionized water, and after uniform dispersion, 1 ml of 0.1% PVP aqueous solution is added to the solution, followed by the addition of 3 ml of 0.1% tetrachloroauric acid solution, and after thorough stirring, 4 ml of ascorbic acid solution (0.01 mol / L) is quickly added. React for 3 hours at room temperature, and after treatment, CdS@Au nanoparticles are obtained.

[0059] Step four, the CdS@Au nanoparticles obtained in step three above were dispersed in 1 ml deionized water, after ultrasonic homogenization, the same volume of FA-PEG-SH solution (containing folic acid (FA) modified polyethylene glycol thiol solution) was added, and the reaction was carried out at room temperature for 24-48 hours in the dark to obtain FA-CdS@Au nanoparticles. The concentration ratio of CdS@Au to FA-PEG-SH solution was 2:1 (1.6 mg / mL: 0.8 mg / mL).

[0060] The preparation method of the Fe3O4@HA nanocapture probe comprises the following steps:

[0061] Step one, 0.36 FeCl3.6H2O, 0.132 g FeCl2.4H2O and 30 mg HA were added to a three-necked flask, and after being stirred uniformly after adding 32.5 ml of deionized water, the reaction was carried out under the protection of nitrogen at 100 degrees Celsius for 15 min.

[0062] Step two, 10 ml of a solution containing 1 g of NaOH was rapidly added to the solution by a syringe, and the reaction was carried out at 100 degrees Celsius for 1 h under the protection of nitrogen.

[0063] Step three, the reaction was transferred to a reaction kettle and aged at 150 degrees Celsius for 24 h. After aging, the solution was loaded into a dialysis bag and dialyzed for 2 days. Fe3O4@HA nanoparticles were obtained. Example 2:

[0064] Characterization of FA-CdS@Au:

[0065] The CdS@Au nanoparticles synthesized in Example 1 were dried and characterized by scanning electron microscopy and transmission electron microscopy. As shown in Figure 2 , the scanning electron microscopy and transmission electron microscopy images show that the synthesized CdS@Au nanoparticles have a regular circular shape, good dispersibility and uniform size. Au nanoparticles are dispersed on the surface of the CdS nanoparticles. The high-resolution electron microscopy image shows that the CdS@Au nanoparticles have two different lattice spacings, 0.355 nm corresponding to the 100 crystal plane of CdS and 0.240 nm corresponding to the 111 crystal plane of Au, as shown in Figure 2 . It can be seen from the element mapping of the CdS@Au nanoparticles that the elements Cd, S and Au are uniformly distributed, as shown in Figure 2 .

[0066] The CdS@Au and CdS nanoparticles synthesized in Example 1 were taken for X-ray diffraction comparative analysis, as shown in Figure 3 , the peaks of the two are consistent with the standard card of CdS and the standard card of Au, which proves the successful synthesis of CdS@Au NPs.

[0067] The CdS@Au NPs synthesized in Example 1 were taken for X-ray photoelectron spectroscopy analysis, as shown in Figure 4 The X-ray photoelectron spectrogram showed that the synthesized CdS@Au existed corresponding copper, sulfur and gold elements. Example 3:

[0068] Feasibility experiment and condition optimization experiment of copper ion replacing cadmium ion to generate CuS@Au nanoparticles

[0069] (1) The CdS@Au synthesized in Example 1 was dispersed in deionized water, and the concentration was quantified. The same volume of copper chloride solution was added to the quantified CdS@Au solution, and the solution was ultrasonically reacted for a certain time, and then the ultraviolet determination was performed, as shown in Figure 5 After the addition of the copper chloride solution, the ultraviolet absorbance of CdS@Au in the range of 300-1100 nm changed. The absorbance of CuS@Au formed after 445 nm was obviously higher than that of CdS@Au.

[0070] (2) The replacement time of copper ion replacing cadmium ion to generate CuS@Au was optimized. The same volume of copper chloride solution was added to the quantified CdS@Au solution, and different groups were reacted in the ultrasonic instrument for different times (1, 5, 10, 20, 30 and 40 min), and the ultraviolet determination of different groups was performed. As shown in Figure 6 The absorbance of CuS@Au at 1064 nm increased with the increase of time, and tended to be stable after 10 min, so the optimal replacement time was selected as 10 min. Example 4:

[0071] Photothermal performance test of CuS@Au generated by replacement:

[0072] The same volume of copper chloride solution was added to the quantified CdS@Au solution, and after ultrasonic reaction for 10 min, the solution was placed under the irradiation of 1064 nm laser with light power density (1.0 W / cm 2 ) for 10 min. The infrared thermal imager was used to collect the temperature change of CuS@Au solution generated by replacement of CdS@Au with different concentrations (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL) under laser irradiation, and the time-temperature change curve was drawn. As shown in Figure 7As shown in the figure, with the increase of the concentration of CdS@Au solution, the generated CuS@Au increases, and the change temperature of the solution gradually increases, and when the concentration of CdS@Au is 200 μg / mL, the change temperature tends to be flat, so the concentration of CdS@Au is selected as 200 μg / mL as the optimal concentration for the subsequent experiment.

[0073] After adding the same volume of copper chloride solution to the quantitative CdS@Au solution and ultrasonic reaction for 10 min, the solution was irradiated under 1064 nm laser with different light power densities (0.5 W / cm 2 , 1.0 W / cm 2 , 1.5 W / cm 2 , 2.0 W / cm 2 , 2.5 W / cm 2 , 3 W / cm 2 ) for 10 min. The infrared thermal imager was used to collect the temperature change of the generated CuS@Au solution under the condition of laser irradiation, and the time-temperature change curve was drawn. As shown in the figure, with the increase of the laser power, the change temperature of the solution gradually increases, and when the laser power density is 2.0 W / cm 2 , the change temperature tends to be flat, so the laser power density is selected as 2.0 W / cm 2 as the optimal laser power density for the subsequent experiment. Figure 8

[0074] (3) In order to detect the photothermal stability of the generated CuS@Au nanomaterial, the same volume of copper chloride solution was added to the CdS@Au (200 μg / mL) solution, and after ultrasonic reaction for 10 min, the replaced solution was irradiated under 1064 nm (laser power density was 2.0 W / cm 2 ) for three cycles to obtain the heating-cooling curve. As shown in the figure, the photothermal stability of the generated CuS@Au is good and is relatively stable under the condition of light irradiation. Figure 9

[0075] Figures 7-10 The result graph of the photothermal performance test of the generated CdS@Au under the irradiation of 1064 nm laser. Example 5:

[0076] The pressure performance test of the generated CuS@Au was carried out:

[0077] ​​Take 20 mg of NH4HCO3 into a gas pressure bottle, add a certain amount of CdS@Au solution (0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL) into an EP tube, then add the same volume of copper chloride solution, ultrasonic reaction for 10 min, transfer the solution in the EP tube to the gas pressure bottle, and place it under the irradiation of 1064 nm laser with light power density (2.0 W / cm 2 ) for 10 min. The pressure change in the bottle is recorded by using a portable pressure gauge, and the time-pressure change curve is drawn. As shown in Figure 11 , under the irradiation of 1064 nm laser, with the increase of the concentration of CdS@Au solution, the generated CuS@Au increases, the change temperature of the solution gradually increases, the gas generated by the decomposition of NH4HCO3 increases, and finally the change pressure in the container increases, and when the concentration of CdS@Au is 200 μg / mL, the increase of the change pressure becomes smaller. Finally, the concentration of 200 μg / mL of CdS@Au is selected as the optimal concentration for testing the pressure performance experiment, which is the same as the optimal concentration obtained by the photothermal experiment.

[0078] In a sealed pressure bottle, add different amounts of NH4HCO3 (20 mg, 40 mg, 60 mg, 80 mg, 100 mg), add a certain amount of CdS@Au solution into an EP tube, then add the same volume of copper chloride solution, ultrasonic reaction for 10 min, transfer the solution in the EP tube to the gas pressure bottle, irradiate under 1064 nm laser with light power density (2.0 W / cm 2 ) for 10 min, and record the pressure change in the bottle by using a portable pressure gauge, and draw the time-pressure change curve. As shown in Figure 12 , after the solution is irradiated for 10 min, the change pressure increases with the increase of the mass of NH4HCO3, and finally the mass of 80 mg of NH4HCO3 is selected as the optimal condition for the experiment.

[0079] Test the ability of the generated CuS@Au to replace NH4HCO3 under different conditions, as shown in Figure 13 , only when CdS@Au, copper chloride solution, 1064 nm laser and ammonium bicarbonate all exist, the change of pressure caused by the decomposition of gas is the largest.

[0080] Figures 11-13 The result graph of the photothermal performance test of the generated CdS@Au under the irradiation of 1064 nm laser. Example 6:

[0081] Characterization of detection probe FA-CdS@Au and capture probe Fe3O4@HA

[0082] (1) The FA-CdS@Au nanoparticles synthesized in Example 1 were dispersed in deionized water, and the quantity was determined. The UV spectrum of the synthesized FA-CdS@Au was compared with that of FA and CdS@Au, as shown in Figure 1, and it can be seen that the FA-CdS@Au exhibits characteristic peaks of FA at 245 nm and characteristic peaks of CdS@Au at 268 nm, respectively, proving the successful synthesis of FA-CdS@Au. Figure 14

[0083] (2) The Fe3O4@HA nanoparticles synthesized in Example 1 were subjected to X-ray diffraction analysis, as shown in Figure 2, and the peaks of Fe3O4@HA were consistent with those of the standard card of Fe3O4, proving the successful synthesis of Fe3O4@HA. Figure 15

[0084] (3) The Fe3O4@HA nanoparticles synthesized in Example 1 were subjected to hydration particle size detection, as shown in Figure 3, and the average particle size of the synthesized Fe3O4@HA was 94 nm, with good dispersibility. Figure 16 Example 7:

[0085] The present application also provides the detection probe FA-CdS@Au and the capture probe Fe3O4@HA prepared in Example 1 for temperature / pressure dual-mode detection of circulating tumor cells (MDA-MB-231).

[0086] First, the feasibility experiment of the detection probe FA-CdS@Au and the capture probe Fe3O4@HA for the instant detection of circulating tumor cells (MDA-MB-231) was carried out:

[0087] A certain concentration of MDA-MB-231 cells was plated in a 12-well plate, and 200 μg / mL of FA-CdS@Au and Fe3O4@HA nanoprobe was added according to different conditions. The well plate was placed on a shaker, shaken for one hour, and then subjected to magnetic separation. After complete magnetic separation, the unbound probe solution was discarded, and the cells were washed three times with PBS solution. Then, 1 ml of PBS was added to each well, mixed uniformly, and then transferred to an EP tube for detection. As shown in Figure 4, under different conditions, only when the detection probe FA-CdS@Au, the capture probe Fe3O4@HA, the cells, and the copper chloride solution provided copper ions, and all four were present, the change in temperature was the highest, indicating that the FA-CdS@Au and Fe3O4@HA nanoprobe can be used for the capture and detection of circulating tumor cells (MDA-MB-231). Figure 17

[0088] The detection probe FA-CdS@Au and the capture probe Fe3O4@HA for temperature / pressure dual-mode detection of circulating tumor cells specifically include the following detection steps:​​​​

[0089] (1) MDA-MB-231 cells were plated in 12-well plates at a concentration of 10,000 cells / mL, 5,000 cells / mL, 1,000 cells / mL, 5000 cells / mL, 1000 cells / mL, 500 cells / mL, 100 cells / mL, 50 cells / mL, and 10 cells / mL;

[0090] (2) The detection probe FA-CdS@Au and the capture probe Fe3O4@HA were added to the cells in step (1) at a concentration of 800 μg / mL and 200 μg / mL, respectively. After administration, the well plate was sealed and placed in a shaker for 30 min to allow the probes to fully bind to the cells. After 30 min of shaking, the bottom of the well plate was attracted by a magnet and the upper liquid was discarded. Then, 1 ml of PBS was added to each well and the liquid was transferred to an EP tube for testing;

[0091] (3) Circulating tumor cells were detected by photothermal method: a 1064 nm laser was used to irradiate the detection solution for 10 min, and an infrared thermal imager was used to collect the temperature change and record the temperature difference;

[0092] (4) Circulating tumor cells were detected by pressure method: 80 mg / mL ammonium bicarbonate was weighed in a sealed pressure bottle, 500 μL of the solution to be tested was added, and a 1064 nm laser was used to irradiate for 10 min. A portable pressure gauge was used to record the pressure change.

[0093] For steps (3) and (4), it should be further noted that Figure 1 (b) The detection probe FA-CdS@Au and the capture probe Fe3O4@HA in the present application are used for temperature / pressure dual-mode detection of circulating tumor cells:

[0094] Circulating tumor cells MDA-MB-231 have phagocyte glycoprotein-1 (CD44) and folate receptor FR on their surface. First, the capture probe Fe3O4@HA can specifically recognize and bind to CD44, and it has magnetic properties. After binding to the cells, it can enrich circulating tumor cells MDA-MB-231 and separate them from other cells by their magnetic properties. At the same time, the detection probe FA-CdS@Au specifically recognizes and binds to FR. After the addition of copper chloride solution, a displacement reaction occurs to generate FA-CuS@Au, which has excellent photothermal properties. Under laser irradiation, FA-CuS@Au can increase the temperature of the solution. On the one hand, it realizes the temperature detection of circulating tumor cells, and on the other hand, the increased temperature can decompose ammonium bicarbonate, realizing the pressure detection of circulating tumor cells.

[0095] The standard curve is plotted with the concentration of MDA-MB-231 cells as the abscissa and the change of temperature of the solution as the ordinate, the regression equation of the standard curve in temperature detection is y=4.3383Logx+1.8415, the correlation coefficient R 2 =0.9958, and the lowest detection limit of temperature detection is calculated to be 6 Cells / mL; the regression equation of the standard curve in pressure detection is y=2.9816Logx+8.2971, the regression coefficient R 2 =0.9922y=4.3383, and the lowest detection limit of pressure detection is calculated to be 5 Cells / mL;

[0096] Figure 18 The result graph of the detection probe FA-CdS@Au and the capture probe Fe3O4@HA for detecting different concentrations of circulating tumor cells under 1064nm laser irradiation through temperature detection. Figure 19 The result graph of the detection probe FA-CdS@Au and the capture probe Fe3O4@HA for detecting different concentrations of circulating tumor cells under 1064nm laser irradiation through pressure detection.

[0097] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement and improvement within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A circulating tumor cell detection kit, characterized by, The FA-CdS@Au nano-detection probe and the Fe3O4@HA nano-capture probe; The copper ion solution is a copper chloride solution, and the mass concentration of the copper chloride solution is 1 mg / mL-2 mg / mL.

2. The circulating tumor cell detection kit as described in claim 1, characterized in that, The mass ratio of the FA-CdS@Au nano-detection probe and the Fe3O4@HA nano-capture probe is 1-4:

1.

3. A method for preparing a circulating tumor cell detection kit, characterized by, The method comprises the following steps: Step one, preparing the FA-CdS@Au nano-detection probe; Step two, preparing the Fe3O4@HA nano-capture probe and the copper ion solution, and the copper ion solution is a copper chloride solution, and the mass concentration of the copper chloride solution is 1 mg / mL-2 mg / mL.

4. The method for preparing the circulating tumor cell detection kit as described in claim 3, characterized in that, The preparation method of the FA-CdS@Au nano-detection probe is as follows: Step 1.1, sequentially adding silica nanoballs, a sodium citrate aqueous solution, a cadmium chloride solution, ammonia water and thiourea into a container, and reacting at 80 DEG C for 3 hours to obtain SiO2@CdS nanoparticles; Step 1.2, adding the SiO2@CdS nanoparticles into a sodium hydroxide solution, removing silica at 90 DEG C for 3 hours to obtain CdS nanoparticles; Step 1.3, adding the CdS nanoparticles into deionized water, and then sequentially adding a polyvinylpyrrolidone solution, a tetrachloroauric acid solution and an ascorbic acid solution, and reacting at room temperature for 3 hours to obtain CdS@Au nanoparticles; Step 1.4, dispersing the CdS@Au nanoparticles in deionized water, and then adding FA-PEG-SH under dark conditions and reacting for 24 hours to obtain the FA-CdS@Au nano-detection probe.

5. The method for preparing the circulating tumor cell detection kit as described in claim 4, characterized in that, The specific steps of step 1.1 are as follows: dispersing silica nanoballs in 100 ml deionized water, ultrasonic dispersing uniformly, and then transferring to a round-bottom flask, adding a sodium citrate aqueous solution into the round-bottom flask and stirring for 5 min, then adding a cadmium chloride solution and stirring again for 5 min, slowly adding 2 ml ammonia water and thiourea, and reacting for 5 min, then transferring the round-bottom flask to an oil bath at 80 DEG C and reacting for 3 hours, and obtaining SiO2@CdS nanoparticles after treatment; wherein the mass / volume ratio of silica nanoballs, deionized water, sodium citrate aqueous solution, cadmium chloride solution, ammonia water and thiourea is 150:100:1:0.5:2:2 mg / ml; wherein the concentrations of the sodium citrate aqueous solution, the cadmium chloride and the thiourea are all 1 mol / L; and the mass concentration of the ammonia water is 28%; In step 1.2, the concentration of the sodium hydroxide solution is 0.5 mol / L; In step 1.3, the mass / volume ratio of the CdS nanoparticles, deionized water, polyvinylpyrrolidone solution, tetrachloroauric acid solution and ascorbic acid solution is 10:10:1:3:4 mg / ml; wherein the mass concentrations of the polyvinylpyrrolidone solution and the tetrachloroauric acid solution are both 0.1%; In step 1.4, the mass concentration ratio of the CdS@Au nanoparticle solution to the FA-PEG-SH solution is 2:1, and the volume ratio is 1:

1.

6. The method for preparing the circulating tumor cell detection kit as described in claim 3, characterized in that, The preparation method of the Fe3O4@HA nano-capture probe is as follows: Step 2.1, FeCl3.6H2O, FeCl2.4H2O and HA were added into a container, then deionized water was added and stirred until uniform, and then heated to 100℃ under reflux for 15 min to obtain a mixed solution; Step 2.2, a solution of NaOH was added to the mixed solution, and reacted at 100℃ for 1h under nitrogen protection to obtain a reaction solution; Step 2.3, the reaction solution was transferred to a reaction kettle, and aged at 150℃ for 24h, and then the solution was loaded into a dialysis bag and dialyzed for 2 days to obtain Fe3O4@HA nanocapture probes.

7. The method for preparing the circulating tumor cell detection kit as described in claim 6, characterized in that, In step 2.1, the mass / volume ratio of FeCl3.6H2O, FeCl2.4H2O, HA and deionized water was 0.36:0.132:30:32.5 g / ml; In step 2.2, the weight ratio of NaOH in the NaOH solution to the mass of HA was 1000:30.

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