CN111454907A - Circulating tumor cell rapid non-invasive capture, release and detection kit - Google Patents

Abstract

A rapid and non-destructive capture, release and detection kit for circulating tumor cells relates to the fields of biotechnology and biomedicine. It includes CTCs magnetic nano-capture probe solution, red blood cell lysate, CTCs release solution, CTCs detection solution, and washing buffer; the CTCs magnetic nano-capture probe is a magnetic nano-particle with folic acid modified on the surface, and the CTCs magnetic nano-capture probe is on the surface of the magnetic nano-particle. The folate specifically binds to the folate receptor on the surface of circulating tumor cells, thereby realizing the capture and separation of circulating tumor cells under an external magnetic field. The CTCs detection solution can realize fluorescent labeling of the released CTCs, which can be observed and counted under a fluorescence microscope. The CTCs captured and released by the kit retains a very high activity, which is of great significance for the early in vitro screening and diagnosis of cancer and the follow-up study of CTCs. The components in the kit can be selectively used according to specific needs, the operation is simple and convenient, and the kit has broad application prospects.

Description

A rapid and non-invasive capture, release and detection kit for circulating tumor cells

technical field

The invention relates to the fields of biotechnology and biomedicine, in particular to a rapid and non-destructive capture, release and detection kit of circulating tumor cells.

Background technique

Circulating tumor cells (CTCs) are important biomarkers in tumor liquid biopsy. CTCs refer to the collective term for tumor cells shed from the primary tumor or metastases into peripheral blood. CTCs appear earlier than solid tumors visible on imaging, and can be used as markers for early diagnosis of tumors. In addition, the number and phenotype of CTCs are closely related to tumor progression, metastasis and prognosis. The clinical detection and counting of CTCs can be used to dynamically monitor the occurrence and development of tumors, evaluate the therapeutic effect of tumors, and guide the formulation and implementation of personalized tumor treatment plans. However, CTCs are extremely rare, and their detection first requires their enrichment.

At present, a series of enrichment and separation methods for CTCs have been developed, which can be roughly divided into two categories according to the principle: enrichment and separation methods based on the physical properties of CTCs (density, size, deformability, dielectric properties, etc.) and biological-based enrichment and separation methods. Affinity recognition enrichment separation method of chemical properties (immunomagnetic separation technology, microfluidic chip technology). Among them, the affinity recognition enrichment separation method based on biochemical properties has been widely used due to its high selectivity and strong specificity. Immunomagnetic separation technology is widely used because of its simple operation, no need for complex instruments, and the ease of combining multiple detection techniques.

In the conventional immunomagnetic separation method, the anti-EpCAM antibody is directly covalently coupled and modified on the surface of the magnetic beads to construct antibody-modified immunomagnetic beads. However, this method has certain limitations: 1) the high cost of antibodies, poor stability, and short shelf life make the cost of immunomagnetic separation relatively high; 2) the process of tumor metastasis to form CTCs will undergo epithelial-mesenchymal transition (EMT) , the epithelial-mesenchymal transition leads to the down-regulation or even non-expression of EpCAM on the cell surface, which makes the EpCAM-based immunomagnetic separation method easy to miss the mesenchymal CTCs; 3) Based on the antigen-antibody recognition method, the magnetic beads will bind to the CTCs 4) Magnetic beads bind to the surface of CTCs, which will eventually be internalized by CTCs, resulting in cytotoxicity and affecting the real properties of CTCs.

At present, the research of CTCs is no longer limited to detection and counting, and it is more meaningful to carry out follow-up research on captured CTCs, such as anti-tumor drug screening, tumor drug resistance research, CTCs genomics research, proteomics research, etc. Wait. Therefore, there is an urgent need for an efficient and rapid CTCs enrichment and separation method without damage, so as to carry out downstream research after CTCs enrichment.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the above problems in the prior art, and to provide a rapid and non-invasive capture, release and detection kit for circulating tumor cells, which provides convenience for the capture, detection and subsequent in-depth research of CTCs.

To achieve the above object, the present invention adopts the following technical solutions:

A kit for rapid and non-destructive capture, release and detection of circulating tumor cells, comprising CTCs magnetic nano-capture probe solution, red blood cell lysate, CTCs release solution, and washing buffer; the CTCs magnetic nano-capture probe is surface-modified with folic acid The folate on the magnetic nanocapture probe of CTCs specifically binds to the folate receptor on the surface of circulating tumor cells, thereby realizing the capture and separation of circulating tumor cells in an external magnetic field.

The present invention also includes a CTCs detection solution, the CTCs detection solution comprises a detection solution A, a detection solution B and a detection solution C; the detection solution A is a 4% paraformaldehyde solution; the detection solution B is a rabbit anti-folate receptor antibody solution ( Rabbit Anti-FRsantibody); solution C is Cy5-labeled goat anti-rabbit antibody solution (Goat-anti Rabbit antibody). The detection solution can realize the fluorescent labeling of CTCs, and realize the detection of CTCs under a fluorescence microscope. Specifically, the CTCs captured by the CTCs magnetic nanocapture probe were separated from the magnetic nanoparticles after being treated with the CTCs release solution, and the CTCs were immobilized on the surface of the glass slide by the detection solution A. The surface exhibits red fluorescence, which can be observed and counted under a fluorescence microscope.

The concentration of the rabbit anti-folate receptor antibody solution is 10 μg/mL, and the concentration of the goat anti-rabbit antibody solution is 10 μg/mL.

The magnetic nanoparticles are carboxyl functionalized magnetic nanoparticles (MNs).

The construction method of the magnetic nanocapture probe for CTCs is as follows: firstly, the disulfide-bonded cystamine molecule (Cystamine) is modified on the surface of the magnetic nanoparticle to form a MNs@Cys conjugated complex, and then the active esterified polymer containing Ethylene glycol chains of folic acid (FA-PEG _2K -NHS) were coupled with the MNs@Cys coupling complex to form MNs@Cys@PEG _2k -FA magnetic nanocapture probes.

The red blood cell lysate contains ammonium chloride.

The CTCs release solution was a cell isotonic buffer containing 50 mM dithiothreitol.

The washing buffer adopts phosphate buffered saline (PBS), which can be used in the washing process of each step.

Compared with the prior art, the beneficial effects obtained by the technical solution of the present invention are:

1. Early diagnosis, intervention and treatment of cancer are effective ways to improve the prognosis and survival rate of cancer patients. The current clinical diagnostic methods still have a certain lag, and it is difficult to achieve early diagnosis. CTCs are considered to be the hallmark of tumorigenesis as an important marker of liquid biopsy. This kit contains a complete set of CTCs enrichment, separation, release and detection components, which can be used for the rapid screening of CTCs in blood, the prognosis of tumor patients, the monitoring of tumor recurrence, and the evaluation kits for individualized treatment of chemotherapy drugs. The formulation of the program is of great significance.

2. The magnetic nanoparticles used in the construction of the CTCs magnetic nano-capture probe in the kit of the present invention are carboxyl-functionalized magnetic nanoparticles, which have fast magnetic response capability and high magnetic recovery efficiency, and can realize the rapid capture and separation of CTCs. .

3. In the kit of the present invention, small molecule folic acid is used instead of the antibody used in the immunomagnetic separation method as the target molecule of CTCs, which has the advantages of low cost, high stability, controllable and easy coupling modification, and long-term storage.

4. In the construction of the magnetic nano-capture probe for CTCs in the kit of the present invention, the cystamine molecule containing disulfide bonds is first modified. Subsequently, folic acid containing polyethylene glycol (PEG) chains was modified on the surface of cystamine molecule-modified magnetic nanoparticles. Due to the steric hindrance of magnetic nanoparticles, folic acid, which is a small molecule directly modified on the surface of magnetic nanoparticles, may not be effectively recognized by the folate receptors on the surface of CTCs due to the steric hindrance effect. The introduction of PEG chains can stretch out folic acid, and the introduction of flexible chains promotes the binding of folic acid on the surface of magnetic nanoparticles to the folic acid receptors on the surface of CTCs, so that the magnetic nanocapture probes of CTCs can be more effectively targeted and bound to the surface of CTCs.

5. The release of magnetically captured CTCs in this kit is achieved by breaking the disulfide bonds on the surface of magnetic nanoparticles, so that the captured and released CTCs have high activity, and can be used for subsequent research, such as detection count, In vitro culture, drug screening, drug resistance analysis, gene sequencing analysis and proteomics research, etc.

6. The CTCs magnetic nano-capture probe constructed in the kit of the present invention is a folic acid-modified CTCs magnetic nano-capture probe, which recognizes and binds to CTCs through the specific binding of folic acid to the folic acid receptor on the surface of CTCs. Since folate receptors are highly expressed in most tumor tissues, the magnetic nanocapture probe for CTCs has universal applicability to a variety of tumor-derived CTCs.

7. This kit is suitable for the capture, release and detection of CTCs in whole blood. The components in the kit can be selectively used according to specific needs.

8. The cost of the kit of the present invention is low, the price is affordable, the kit method is relatively simple to use, easy to operate, and saves time and effort.

9. The red blood cell lysing solution can effectively lyse red blood cells in whole blood, thereby reducing the complexity of blood.

Description of drawings

FIG. 1 is a schematic diagram of the capture and release of CTCs involved in the kit of the present invention and a schematic diagram of subsequent applications.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more comprehensible, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

A kit for rapid and non-invasive capture, release and detection of circulating tumor cells, comprising CTCs magnetic nano-capture probe solution, red blood cell lysate, CTCs release solution, CTCs detection solution, and washing buffer;

The red blood cell lysing solution is composed of ammonium chloride, which is used for lysing red blood cells in whole blood; the washing buffer adopts phosphate buffered saline (PBS); the CTCs detection solution comprises detection solution A, detection solution B and detection solution Solution C; detection solution A is 4% paraformaldehyde solution; detection solution B is rabbit anti-folate receptor antibody solution 10 μg/mL; solution C is Cy5-labeled goat anti-rabbit antibody solution 10 μg/mL.

The CTCs magnetic nano-capture probe is a magnetic nanoparticle with folic acid modified on the surface, and the folic acid on the CTCs magnetic nano-capture probe specifically binds to the folic acid receptor on the surface of circulating tumor cells, thereby realizing circulating tumor cells in an external magnetic field. The CTCs were separated from the magnetic nanoparticles after being treated with the CTCs release solution. The detection solution A immobilized the CTCs on the surface of the glass slide. After being treated with the detection solution B and the detection solution C, the surface of the CTCs showed red fluorescence. Counts can be observed under a fluorescence microscope.

The construction method of the CTCs magnetic nano-capture probe is as follows:

First, the disulfide-bonded cystamine molecule (Cystamine) was modified on the surface of the magnetic nanoparticles to form the MNs@Cys conjugated complex, and then the active esterified polyethylene glycol chain-containing folic acid (FA-PEG _2K -NHS ) was coupled with the MNs@Cys coupling complex to form the MNs@Cys@PEG _2k -FA magnetic nanocapture probe. Specifically, the magnetic nanoparticles are carboxyl-functionalized magnetic nanoparticles (MNs), and the magnetic nanoparticles of the present invention are carboxyl-functionalized ferric oxide nanoparticles.

The CTCs release solution can break the disulfide bond in the cystamine (Cys) in the folic acid-modified magnetic nanoprobe, so that the CTCs magnetic nanocapture probe falls off the surface of the CTCs, and realizes the non-destructive release of the CTCs after capture;

In this example, CTCs were released as a cell isotonic buffer containing 50 mM dithiothreitol, which contained various nutrients required for cell growth. Its main components are: 200mg/L CaCl ₂ , 97.67mg/L MgSO ₄ , 400mg/L KCl, 3700mg/L NaHCO ₃ , 6400mg/L NaCl, 30mg/L glycine, 84mg/L L-arginine hydrochloride , 62.6mg/L L-cystine dihydrochloride, 584mg/L L-glutamine, 42mg/L L-histidine hydrochloride monohydrate, 105mg/L L-isoleucine, 105mg /L L-leucine, 146mg/L L-lysine hydrochloride, 30mg/L L-methionine, 66mg/L L-phenylalanine, 42mg/L L-serine, 95mg/L L-threonine Amino acid, 16mg/L L-tryptophan, 103.79mg/L L-tyrosine disodium salt dihydrate, 94mg/L L-valine, 4mg/L choline chloride, calcium D-pantothenate , folic acid, niacinamide, pyridoxine hydrochloride and thiamine hydrochloride, 0.4mg/L riboflavin, 7.2mg/L meso-inositol, 4500mg/L D-glucose, 5958mg/L HEPES, 15mg/L phenol red, 100kU penicillin G sodium salt and 100mg/L streptomycin sulfate.

As shown in Figure 1, the principle of the present invention is as follows:

1. The folic acid-modified CTCs magnetic nano-capture probe in the kit can recognize and bind to the surface folate receptors of CTCs, so that the CTCs magnetic nano-capture probe can be targeted and bound to the surface of CTCs. The capture and enrichment of CTCs can be achieved in the rack. The magnetic nanocapture probe for CTCs constructed in the kit contains cystamine molecules, which contain disulfide bonds, and the CTCs release solution contains the reducing agent DTT. The opening of the sulfur bond destroys the integrity of the magnetic capture probe. At this time, the chain connecting the folic acid and the magnetic nanoparticles is broken, and the magnetic nanoparticles will fall off from the CTCs to achieve the non-invasive release of the captured cells.

2. The release solution also contains various nutrients required for cell growth, which provides an isotonic environment for CTCs and various components required for cell growth, which facilitates subsequent research or application of CTCs.

3. After the released cells are fixed with detection solution A, the released CTCs can be fluorescently labeled by staining with detection solutions B and C respectively, which can be observed and counted under a fluorescence microscope. The CTCs captured and released by the kit retains a very high activity, which is of great significance for the early in vitro screening and diagnosis of cancer and the follow-up study of CTCs. The components in the kit can be selectively used according to specific needs, the operation is simple and convenient, and the kit has broad application prospects.

In the present invention, the coupling preparation of the folic acid-modified CTCs magnetic nano-capture probe includes the following steps:

(1) Take 1 mg of magnetic nanoparticles and resuspend in 1 mL of 25 mM MES buffer (pH 6.0), and magnetically separate and wash for 3 times;

(2) Resuspend the magnetic nanoparticles in 1mL 25mM MES buffer in a 2mL centrifuge tube, add 29.5μg 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC·HCl) and 32.6 μg of N,N-hydroxysuccinimide ester (Sulfo-NHS) (EDC·HCl and Sulfo-NHS solutions need to be prepared and used immediately), placed on a rotary mixer and incubated at room temperature for 45 minutes;

(3) Remove the clear night by magnetic separation, remove excess EDC·HCl/Sulfo-NHS, wash twice with PBS, resuspend in 1 mL PBS solution (1×PBS, pH 7.4), add 0.1 mg of cystamine hydrochloride, After mixing, place it on a rotary mixer and continue to mix and incubate for 3h at room temperature;

(4) Add 50 μg of ethanolamine and incubate for 1 h to block the uncoupled activated carboxyl groups;

(5) Magnetic separation to remove the clear night, the conjugated complex was washed twice with PBS and resuspended in 1 mL of PBS solution;

(6) Add 1.5 mg of FA-PEG _2k -NHS, mix evenly, place it on a rotary mixer and continue to mix and incubate for 3h at room temperature;

(7) The conjugated complex was washed with PBS and then resuspended in 1 mL of PBS solution to obtain the CTCs magnetic nanocapture probe solution.

Example 1

The enrichment and separation of CTCs in cell suspension includes the following steps:

(1) When HEK293 cells are cultured to 70% to 80% confluence, they are digested with trypsin, pipetting and mixing to prepare a single cell suspension (the concentration of the cell suspension is 10 ⁶ /mL, simulating monocytes in blood), take 1 mL Add to 2mL bovine serum albumin-coated centrifuge tube;

(2) Different numbers of pre-stained cervical cancer cells (Hela) were added to HEK293 cell suspension to simulate CTCs;

(3) After mixing the cell suspension, add 100 μL of folic acid-modified CTCs magnetic nanocapture probe solution, place it on a rotary mixer and incubate for 15 minutes at room temperature;

(4) Then the centrifuge tube was placed in a 0.6T magnetic rack, magnetically separated for 2 min, and the supernatant was discarded;

(5) 100 μL of PBS containing 1% BSA to resuspend the magnetic nanoprobe-CTCs complex;

(6) Spread the resuspension on a glass slide and place it under a fluorescence microscope to observe and count;

(7) Calculation of capture efficiency: capture efficiency=[number of magnetically separated cancer cells/number of labeled cancer cells]*100%.

The capture efficiency of each group is shown in Table 1, as follows:

Table 1

number of marks twenty three 43 112 141 204 257 290 Number of catches twenty one 39 107 129 197 236 266 Capture efficiency 91.3% 90.7% 87.7% 91.5% 96.6% 91.8% 91.7%

The experimental results show that the folic acid-modified CTCs magnetic nanocapture probe can efficiently enrich target tumor cells from complex background cells. Adding different concentrations of tumor cells to the cell suspension simulates the number of tumor cells in the peripheral blood of cancer patients with different stages of progression.

Example 2

The capture of CTCs in blood includes the following steps:

(1) Add 1 mL of blood to a 10 mL centrifuge tube, add pre-stained cervical cancer cells (Hela) to the blood to simulate CTCs, and mix by pipetting gently;

(2) Add 3 mL of erythrocyte lysate to the centrifuge tube, let the centrifuge tube stand for 10 minutes, and shake several times during this period;

(3) After mixing the cell suspension, add 100 μL of folic acid-modified CTCs magnetic nanocapture probe, and place it on a rotary mixer for continuous incubation at room temperature for 15 minutes;

(4) After the lysis, 2000rpm, 3min, suction and discard the supernatant, resuspend the precipitate in 1mL of PBS, and transfer it to a 2mL BSA-coated centrifuge tube;

(5) Add 100 μL of folic acid-modified CTCs magnetic nano-capture probe solution to it, and place it on a rotary mixer for continuous incubation at room temperature for 15 min;

(6) Then the centrifuge tube was placed in a 0.6T magnetic rack, magnetically separated for 2min, and the supernatant was discarded;

(7) 100 μL of PBS containing 1% BSA to resuspend the magnetic nanoprobe-CTCs complex;

(8) Spread the resuspension on a glass slide and place it under a fluorescence microscope to observe and count;

(9) Calculation of capture efficiency: capture efficiency=[number of magnetically separated cancer cells/number of labeled cancer cells]*100%.

The capture efficiency is shown in Table 2:

Table 2

The experimental results show that the magnetic nanocapture probe for CTCs can achieve efficient and rapid capture of CTCs in blood, and can be applied to the rapid capture of CTCs in blood samples of clinical tumor patients.

Example 3

The verification of the release efficiency of magnetically captured CTCs includes the following steps:

(1) In this example, about 200 pre-stained HeLa cells were added to 1 mL of HEK293 cell suspension, the centrifuge tube was inverted for several times, and then 100 μL of the CTCs magnetic nanocapture probe solution was added. Incubate at room temperature for 15 min;

(2) Then the centrifuge tube was placed in a 0.6T magnetic rack, magnetically separated for 2 min, and the supernatant was discarded;

(3) 1 mL of CTCs-containing release solution to resuspend the magnetic nanoprobe-CTCs complex should be placed in a constant temperature water bath at 37°C for continuous incubation for 30 min;

(4) Then the centrifuge tube was placed in a 0.6T magnetic rack, magnetically separated for 2 min, and the supernatant was discarded;

(5) 100 μL of PBS containing 1% BSA to resuspend the magnetic nanoprobe-CTCs complex;

(6) Spread the resuspension on a glass slide and place it under a fluorescence microscope to observe and count.

(7) Calculation of release efficiency: release efficiency=[1-(the number of cancer cells in the second magnetic enrichment/the number of cancer cells in the first magnetic enrichment)]*100%.

The release efficiency results are shown in Table 3:

table 3

Sample number/DTT processing time 30min 45min 1 81.6% 77.7% 2 79.7% 83.1 3 76.3% 85% average value 79.2% 81.9%

The experimental results show that the magnetic nanoparticles can realize the capture and release of CTCs. After the CTCs release solution was treated at 37°C for 30 min, the release efficiency of magnetically captured CTCs was close to 80%.

Example 4

Activity verification of the released CTCs, including the following steps:

(1) In this example, Hela cells were digested with trypsin, pipetted into a single-cell suspension, and diluted to 10 ³ -10 ⁴ /mL with cell culture medium;

(2) Take 1 mL of the cell suspension, add 100 μL of the CTCs magnetic nanocapture probe solution, and place it on a rotary mixer for continuous incubation at room temperature for 15 min;

(3) Then the centrifuge tube was placed in a 0.6T magnetic rack, magnetically separated for 2 min, and the supernatant was discarded;

(4) The magnetic nanoprobe-CTCs complex was resuspended in 1 mL of CTCs release solution and incubated in a constant temperature water bath at 37°C for 30 min;

(5) Then the centrifuge tube was placed in a 0.6T magnetic frame, magnetically separated for 2min, and the supernatant was collected;

(6) 1500rmp, 3min, suction and discard the supernatant, and collect the cell pellet;

(7) Resuspend cells in 100 μL of PBS, add 2 μL of 1 mM Calcein-AM and 3 μL of 1.5 mM PI (propidium iodide), mix and incubate at 37°C for 15 min;

(8) After washing twice with PBS, the resuspension was spread on a glass slide and placed under a fluorescence microscope to observe, photograph and count.

Live/Dead Cell Staining Results:

The experimental results found that the released Hela cells basically showed green fluorescence, and only a few cells showed red fluorescence, indicating that the released cells still had high activity. According to the statistical analysis of Image J software, the content of live cells with green fluorescence accounted for 98.6%, indicating that the kit in the invention can achieve the non-destructive capture and release of CTCs.

Example 5

The in vitro culture of the released CTCs includes the following steps:

(1) In this example, Hela cells were digested with trypsin, pipetted into a single-cell suspension, and diluted to 10 ³ -10 ⁴ /mL with cell culture medium;

(2) Take 1 mL of the cell suspension, add 100 μL of the CTCs magnetic nanocapture probe solution, and place it on a rotary mixer for continuous incubation at room temperature for 15 min;

(3) Then the centrifuge tube was placed in a 0.6T magnetic rack, magnetically separated for 2 min, and the supernatant was discarded;

(4) The magnetic nanoprobe-CTCs complex was resuspended in 1 mL of CTCs release solution and incubated in a constant temperature water bath at 37°C for 30 min;

(5) Then the centrifuge tube was placed in a 0.6T magnetic frame, magnetically separated for 2min, and the supernatant was collected;

(6) 1500rmp, 3min, suction and discard the supernatant, and collect the cell pellet;

(7) Resuspend the cells in 1 mL of cell culture medium, transfer them to a 6-well plate and place them in a constant temperature and humidity incubator for cultivation.

In vitro culture results:

The experimental results showed that the cells adhered well and almost no dead cells were observed after culturing for 24 hours. Some cells have begun to divide and proliferate. Subsequently, the cells were cultured in vitro for 48h and 72h to observe the growth of the cells. The growth and proliferation of the cells were normal, and there was no significant difference in the morphology of the cells from the control group (without any treatment). The folic acid-modified CTCs magnetic nanocapture probe can realize the efficient and non-invasive separation of CTCs, and the released CTCs maintain high cell activity, which can be directly cultured in vitro, which provides the possibility for the downstream research of CTCs.

Example 6

The proteomic application of CTCs cultured in vitro includes the following steps:

(1) Take the tumor cells cultured in vitro for 72 hours after release, wash 3 times with cold PBS, scrape the adherent growing cells in the culture dish with a cell scraper, transfer them to a centrifuge tube, and centrifuge at 800g for 5min to collect the cell pellets in 1.5mL in a low adsorption centrifuge tube;

(2) 0.5mL of cell lysate (100mM Tris-HCl, 8M urea, 150mM NaCl, 1×protease inhibitors cocktial, 1×phosphatase inhibitors cocktial, 1mM sodium vanadate, 1mM PMSF) was added to a 1.5mL centrifuge tube, 4°C Let stand for 10min cracking;

(3) Ultrasonic treatment of lysate, ultrasonic for 5s, pause for 25s, ultrasonic for 1min;

(4) Centrifuge at 12000g for 10min, collect the supernatant in a new 1.5mL ultrafiltration centrifuge tube (10KD);

(5) Centrifuge at 3000g, 14°C for 30min, repeat twice;

(6) Add TCEP to make the final concentration 30mM, after incubation at room temperature for 10min, add iodoacetamide (IAA) solution to make the final concentration 50mM, and react at room temperature for 30min in the dark;

(7) at 3000g, 14°C, centrifuged for several times, and replaced with ammonium bicarbonate solution, so that the urea concentration is lower than 0.5M;

(8) Collect the protein solution in the ultrafiltration tube, after measuring the protein concentration by Bradford method, add mass spectrometry-grade trypsin at the ratio of protein:trypsin=50:1, and digest overnight;

(9) add 10% formic acid solution to the enzyme digestion solution, make its final concentration be 5%, use the C18 desalting column of Waters to remove salt;

(10) 50% acetonitrile was used to elute the peptide segment in the desalting column, and it was dried;

(11) After dissolving the peptide fragments in 0.1% formic acid solution, the mass spectrometry detection analysis (LC-MS/MS) was performed after the concentration was tested.

Mass spectrometry identification results:

Proteomics can provide key protein information about the cellular heterogeneity, disease stage, and survival chances of CTCs. The non-invasive separation and release of CTCs provides the possibility for the proteomic study of CTCs. Based on the enrichment and release method of this kit, the model CTCs cultured in vitro after release were identified by proteomics, and a total of 3754 proteins were identified in the experiment.

Example 7

The detection of CTCs in blood includes the following steps:

(1) Add 1 mL of blood to a 10 mL centrifuge tube, add pre-stained cervical cancer cells (Hela) to the blood to simulate CTCs, and mix by pipetting gently;

(2) Add 3 mL of erythrocyte lysate to the centrifuge tube, let the centrifuge tube stand for 10 minutes, and shake several times during this period;

(3) After mixing the cell suspension, add 100 μL of folic acid-modified CTCs magnetic nanocapture probe, and place it on a rotary mixer for continuous incubation at room temperature for 15 minutes;

(4) After the lysis, 2000rpm, 3min, suction and discard the supernatant, resuspend the precipitate in 1mL of PBS, and transfer it to a 2mL BSA-coated centrifuge tube;

(5) Add 100 μL of folic acid-modified CTCs magnetic nano-capture probe to it, place it on a rotary mixer and incubate for 15 min at room temperature;

(4) Then the centrifuge tube was placed in a 0.6T magnetic rack, magnetically separated for 2 min, and the supernatant was discarded;

(5) 1 mL of CTCs release solution to resuspend the magnetic nanoprobe-CTCs complex should be placed in a constant temperature water bath at 37°C for continuous incubation for 30 min;

(6) Then the centrifuge tube was placed in a 0.6T magnetic rack, magnetically separated for 2min, and the supernatant was discarded;

(7) 200 μL of 4% paraformaldehyde solution was resuspended and then smeared on the confocal culture dish, and fixed at room temperature for 30 minutes;

(8) After washing three times with PBS, block with 5% BSA for 1 h at room temperature;

(9) After washing 3 times with PBS, add 1 mL of detection solution B, and incubate at room temperature for 1 h;

(10) After washing 3 times with PBS, add 1 mL of detection solution C, and let stand for 30 min at room temperature;

(11) After washing three times with PBS, the staining results of cells were observed under a laser confocal microscope.

The results of cell staining showed that the tumor cells showed red fluorescence, the nucleus was blue and the cell size was larger than 8 μm, which was obviously different from blood cells. It shows that this kit can be used for the detection of CTCs in blood.

Claims (8)

1. A circulating tumor cell rapid non-invasive capture, release and detection kit is characterized in that: comprises CTCs magnetic nano capture probe solution, erythrocyte lysate, CTCs release solution and washing buffer solution; the CTCs magnetic nano capture probe is a magnetic nano particle with the surface modified with folic acid, and folic acid on the CTCs magnetic nano capture probe is specifically combined with a folic acid receptor on the surface of the circulating tumor cell, so that the circulating tumor cell is captured and separated on an external magnetic field.

2. The kit for rapid non-invasive capture, release and detection of circulating tumor cells according to claim 1, wherein: the kit also comprises CTCs detection liquid, wherein the CTCs detection liquid comprises detection liquid A, detection liquid B and detection liquid C; the detection solution A is a 4% paraformaldehyde solution; the detection solution B is a rabbit anti-folate receptor antibody solution; the solution C is Cy5 labeled goat anti-rabbit antibody solution; the CTCs captured by the CTCs magnetic nano capture probe are separated from the magnetic nanoparticles after being treated by CTCs release liquid, the CTCs are fixed on the surface of the glass slide by detection liquid A, and after being respectively treated by detection liquid B and detection liquid C, the surface of the CTCs shows red fluorescence which can be observed and counted under a fluorescence microscope.

3. The kit for rapid non-invasive capture, release and detection of circulating tumor cells according to claim 1, wherein: the magnetic nanoparticles are carboxyl-functionalized Magnetic Nanoparticles (MNs).

4. The kit for rapid non-invasive capture, release and detection of circulating tumor cells according to claim 3, wherein the CTCs magnetic nano capture probe is constructed by the following method: firstly, Cystamine molecules (Cystamine) with disulfide bonds are modified on the surface of magnetic nanoparticles to form MNs @ Cys coupling compound, and then active esterified folic acid (FA-PEG) containing polyethylene glycol chains_2K-NHS) with an MNs @ Cys conjugate complex to form MNs @ Cys @ PEG_2k-FA magnetic nano-capture probe.

5. The kit for rapid non-invasive capture, release and detection of circulating tumor cells according to claim 1, wherein: the erythrocyte lysate contains ammonium chloride.

6. The kit for rapid non-invasive capture, release and detection of circulating tumor cells according to claim 1, wherein: the CTCs release solution is a cell isotonic buffer solution containing 50mM dithiothreitol.

7. The kit for rapid non-invasive capture, release and detection of circulating tumor cells according to claim 1, wherein: the washing buffer adopts phosphate buffer.

8. The kit for rapid non-invasive capture, release and detection of circulating tumor cells according to claim 2, wherein the concentration of said rabbit anti-folate receptor antibody solution is 10 μ g/m L, and the concentration of said goat anti-rabbit antibody solution is 10 μ g/m L.

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