CN111454907A - Circulating tumor cell rapid non-invasive capture, release and detection kit - Google Patents
Circulating tumor cell rapid non-invasive capture, release and detection kit Download PDFInfo
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Abstract
A kit for rapid and nondestructive capture, release and detection of circulating tumor cells relates to the field of biotechnology and biomedicine. Comprises CTCs magnetic nano capture probe solution, erythrocyte lysate, CTCs release solution, CTCs detection 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. The CTCs detection solution can realize fluorescent labeling on the released CTCs, and the count is observed under a fluorescent microscope. The kit can capture and release CTCs and keep very high activity, and has great significance for early in-vitro screening diagnosis of cancers and subsequent research of CTCs. The components in the kit can be selectively used according to specific requirements, and the kit is simple and convenient to operate and has a wide application prospect.
Description
Technical Field
The invention relates to the field of biotechnology and biomedicine, in particular to a kit for rapidly capturing, releasing and detecting circulating tumor cells without damage.
Background
Circulating Tumor Cells (CTCs) are important biomarkers for tumor fluid biopsy. CTCs are a generic term for tumor cells that have been shed from the primary foci or metastases of the tumor into the peripheral blood. CTCs appear earlier than solid tumors visible by 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 for dynamic monitoring of tumorigenesis and development, evaluation of tumor treatment effect and guidance of preparation and implementation of tumor personalized treatment schemes. However, the content of CTCs is extremely rare and its detection requires first enrichment.
At present, a series of enrichment and separation methods for CTCs have been developed, and are roughly divided into two main categories according to the principle: an enrichment and separation method based on the physical properties of CTCs (density, size, deformability, dielectricity and the like) and an affinity identification and enrichment and separation method based on biochemical properties (immunomagnetic separation technology and microfluidic chip technology). Among them, the affinity recognition, enrichment and separation method based on biochemical properties has been widely used due to high selectivity and strong specificity. The immunomagnetic separation technology is easy to operate, does not need complex instruments and equipment, and is easy to combine multiple detection technologies, so that the immunomagnetic separation technology can be widely applied.
In a conventional immunomagnetic separation method, an anti-EpCAM antibody is directly covalently coupled and modified on the surface of a magnetic bead to construct an antibody-modified immunomagnetic bead. However, this approach has certain limitations: 1) the antibody has high cost, poor stability and short shelf life, so that the immunomagnetic separation cost is higher; 2) the tumor metastasis process of forming CTCs can undergo an epithelial-mesenchymal transition process (EMT), and the epithelial-mesenchymal transition causes the expression of EpCAM on the cell surface to be reduced or even not expressed, so that the EpCAM-based immunomagnetic separation method is easy to miss detection of mesenchymal CTCs; 3) based on an antigen-antibody recognition mode, magnetic beads can be combined on the surfaces of CTCs to influence fluorescent labeling and detection; 4) the magnetic beads are bonded on the surfaces of the CTCs and are finally internalized by the CTCs, so that cytotoxicity is generated, and the real properties of the CTCs are influenced.
At present, the research on the CTCs is not limited to detection and counting any more, and the subsequent research on the captured CTCs is more significant, such as screening of anti-tumor drugs, research on drug resistance of tumors, research on genomics and proteomics of the CTCs, and the like. Therefore, an efficient and rapid nondestructive enrichment and separation method for CTCs is urgently needed to develop downstream research after the enrichment of CTCs.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides a kit for rapidly and nondestructively capturing, releasing and detecting circulating tumor cells, which is convenient for capturing and detecting CTCs and further researching the CTCs.
In order to achieve the purpose, the invention adopts the following technical scheme:
a kit for rapid and nondestructive capture, release and detection of circulating tumor cells 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.
The invention 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 (Rabbit Anti-FRsantobidy); the solution C is Cy5 labeled Goat anti-Rabbit antibody solution (Goat-anti Rabbit antibody). The detection liquid can realize the fluorescent labeling of the CTCs and the detection of the CTCs under a fluorescent microscope. Specifically, the CTCs captured by the CTCs magnetic nano capture probe are separated from the magnetic nanoparticles after being treated by CTCs release solution, the CTCs are fixed on the surface of the glass slide by detection solution A, and after being respectively treated by detection solution B and detection solution C, the surface of the CTCs shows red fluorescence, and can be observed and counted under a fluorescence microscope.
The concentration of the rabbit anti-folate receptor antibody solution is 10 mu g/m L, and the concentration of the goat anti-rabbit antibody solution is 10 mu g/m L.
The magnetic nanoparticles are carboxyl-functionalized Magnetic Nanoparticles (MNs).
The construction method of the CTCs magnetic nano capture probe comprises the following steps: 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 chains2K-NHS) with an MNs @ Cys conjugate complex to form MNs @ Cys @ PEG2k-FA magnetic nano-capture probe.
The erythrocyte lysate contains ammonium chloride.
The CTCs release solution is a cell isotonic buffer solution containing 50mM dithiothreitol.
The washing buffer solution adopts Phosphate Buffer Solution (PBS), and can be used in the washing process of each step.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. early diagnosis, intervention and treatment of cancer is an effective way to improve prognosis and survival of cancer patients. The current clinical diagnosis method still has certain hysteresis and is difficult to realize early diagnosis. CTCs are considered as important markers of tumorigenesis as markers of fluid biopsy. The kit comprises a whole set of CTCs enriching, separating, releasing, detecting and other components, can be used for rapidly screening CTCs in blood, and has important significance for prognosis of tumor patients, monitoring of tumor recurrence, formulation of individualized treatment schemes of evaluation boxes of chemotherapeutic drug effects and the like.
2. The magnetic nanoparticles used for constructing the magnetic nanometer capture probes of the CTCs in the kit are carboxyl functionalized magnetic nanoparticles, have quick magnetic response capability and higher magnetic recovery efficiency, and can realize quick capture and separation of the CTCs.
3. The kit provided by the invention adopts the small molecular folic acid to replace an antibody used in an immunomagnetic separation method as a targeting molecule of CTCs, and has the advantages of low cost, high stability, controllability, easiness in coupling modification, long-term storage and the like.
4. In the construction of the CTCs magnetic nano capture probe in the kit, cystamine molecules containing disulfide bonds are firstly modified. Subsequently, folic acid containing polyethylene glycol (PEG) chains is modified on the surface of the magnetic nanoparticles modified by cystamine molecules. Because the magnetic nanoparticles have certain steric hindrance, folic acid which is a small molecule directly modified on the surfaces of the magnetic nanoparticles may not be effectively recognized by folic acid receptors on the surfaces of CTCs due to the steric hindrance effect. The introduction of the PEG chain can stretch folic acid out, and the introduction of the flexible chain promotes the folic acid on the surface of the magnetic nanoparticles to be combined with folic acid receptors on the surfaces of CTCs, so that the magnetic nano-capture probes of the CTCs are more effectively and targetedly combined to the surfaces of the CTCs.
5. The release of the magnetically-captured CTCs in the kit is realized by breaking the disulfide bonds on the surfaces of the magnetic nanoparticles, so that the captured and released CTCs have higher activity and can be subjected to subsequent researches such as detection and counting, in-vitro culture, drug screening, drug resistance analysis, gene sequencing analysis, proteomics research and the like.
6. The CTCs magnetic nano-capture probe constructed in the kit is a folic acid modified CTCs magnetic nano-capture probe, and the recognition and combination with CTCs are realized by specifically combining folic acid with folic acid receptors on the surfaces of CTCs. As most tumor tissues highly express folate receptors, the magnetic nano-capture probes for CTCs have universal applicability to CTCs derived from various tumors.
7. The kit is suitable for capturing, releasing, detecting and the like of CTCs in whole blood, and each component in the kit can be selectively used according to specific requirements.
8. The kit has the advantages of low cost, low price, relatively simple use of the kit method, easy operation, time saving and labor saving.
9. The erythrocyte lysate can effectively lyse erythrocytes in whole blood, thereby reducing the complexity of blood.
Drawings
FIG. 1 is a schematic diagram of the capture and release of CTCs involved in the kit of the present invention and subsequent application.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.
A kit for rapid and nondestructive capture, release and detection of circulating tumor cells comprises CTCs magnetic nano capture probe solution, erythrocyte lysate, CTCs release solution, CTCs detection solution and washing buffer solution;
the erythrocyte lysate is ammonium chloride and is used for lysing erythrocytes in whole blood, the washing buffer solution is Phosphate Buffered Saline (PBS), 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 10 mu g/m L, and the solution C is a Cy5 labeled goat anti-rabbit antibody solution 10 mu g/m L.
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; and (3) the CTCs are separated from the magnetic nanoparticles after being treated by the CTCs release solution, the CTCs are fixed on the surface of the glass slide by the detection solution A, and the surface of the CTCs shows red fluorescence after being treated by the detection solution B and the detection solution C respectively, so that the CTCs can be observed and counted under a fluorescence microscope.
The construction method of the CTCs magnetic nano capture probe comprises the following steps:
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 chains2K-NHS) with an MNs @ Cys conjugate complex to form MNs @ Cys @ PEG2k-FA magnetic nano-capture probe. Specifically, the magnetic nanoparticles are carboxyl-functionalized Magnetic Nanoparticles (MNs), and the magnetic nanoparticles are carboxyl-functionalized ferroferric oxide nanoparticles.
The CTCs release solution can break disulfide bonds in cystamine (Cys) in the folic acid modified magnetic nanoprobe, so that the CTCs magnetic nanoprobe falls off from the surface of CTCs, and nondestructive release of the CTCs after capture is realized;
in this example, CTCs were released as a cell-isotonic buffer containing 50mM dithiothreitol, containing various nutrients necessary for cell growth, and had a main composition of 200 mg/L CaCl2，97.67mg/L MgSO4,400mg/L KCl，3700mg/L NaHCO36400 mg/L NaCl, 30 mg/L glycine, 84 mg/L0L 1-arginine hydrochloride, 62.6 mg/L2L 3-cystine dihydrochloride, 584 mg/L4L 5-glutamine, 42 mg/L6L 7-histidine hydrochloride monohydrate, 105 mg/L8L 9-isoleucine, 105 mg/L L0-leucine, 146 mg/L1L 2-lysine hydrochloride, 30 mg/L3L 4-methionine, 66 mg/L5L 6-phenylalanine, 42 mg/L7L 8-serine, 95 mg/L9L-threonine, 16 mg/L0L 1-tryptophan, 103.79 mg/103.79 2 103.79-tyrosine disodium salt dihydrate, 94 mg/103.79 4 103.79-valine, 4 mg/103.79 choline chloride, D-nicotinamide, calcium pantothenate, pyridoxamine, 103.79G/103.79-tyrosine disodium salt dihydrate, 4500 mg/103.79-calcium chloride, 103.79G/103.79-glucose hydrochloride, 4500 mg/103.79-calcium chloride, 103.79G/103.79-methionine hydrochloride, and 4500 mg/103.79-methionine hydrochloride.
As shown in fig. 1, the principle of the present invention is as follows:
1. the folic acid modified magnetic nano-capture probes of the CTCs in the kit can be identified and combined with surface folate receptors of the CTCs, so that the magnetic nano-capture probes of the CTCs are combined to the surfaces of the CTCs in a targeted manner, and the CTCs can be captured and enriched in an external magnetic field or a magnetic frame. The CTCs magnetic nano-capture probe constructed in the kit contains cystamine molecules, the cystamine molecules contain disulfide bonds, CTCs release solution contains a reducing agent DTT component, the DTT can reduce the disulfide bonds in the cystamine molecules so as to open the disulfide bonds and destroy the integrity of the magnetic capture probe, at the moment, chains connecting folic acid and the magnetic nano-particles are broken, and the magnetic nano-particles fall off from the CTCs to realize the nondestructive release of captured cells.
2. The release liquid also contains various nutrient components required by cell growth, provides an isotonic environment for the CTCs and various components required by cell growth, and is convenient for developing subsequent research or application of the CTCs.
3. After the released cells are fixed by the detection liquid A, the released CTCs can be subjected to fluorescence labeling by staining with the detection liquid B and staining with the detection liquid C respectively, and the released CTCs can be observed and counted under a fluorescence microscope. The kit can capture and release CTCs and keep very high activity, and has great significance for early in-vitro screening diagnosis of cancers and subsequent research of CTCs. The components in the kit can be selectively used according to specific requirements, and the kit is simple and convenient to operate and has a wide application prospect.
In the invention, the coupled preparation of the folic acid modified CTCs magnetic nano capture probe comprises the following steps:
(1) 1mg of magnetic nanoparticles was resuspended in 1m L of 25mM MES buffer (pH 6.0) and washed 3 times by magnetic separation;
(2)1m L25 mM MES buffer resuspend the magnetic nanoparticles in a 2m L centrifuge tube, add 29.5. mu.g of 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride (EDC. HCl) and 32.6. mu. g N, N-hydroxysuccinimide ester (Sulfo-NHS) (EDC. HCl and Sulfo-NHS solutions are ready for use), place on a rotary mixer and incubate for 45min with constant mixing at room temperature;
(3) removing supernatant by magnetic separation, removing excessive EDC & HCl/Sulfo-NHS, washing with PBS twice, suspending in 1m L PBS solution (1 × PBS, pH 7.4), adding 0.1mg cystamine hydrochloride, mixing, placing on a rotary mixer, and incubating for 3h at room temperature;
(4) adding 50 mu g of ethanolamine to incubate for 1h, and blocking the unconjugated activated carboxyl;
(5) the supernatant was removed by magnetic separation, and the conjugate complex was washed twice with PBS and resuspended in 1m L PBS solution;
(6) adding 1.5mg of FA-PEG2k-NHS, mixing evenly, placing on a rotary mixer, and continuously mixing and incubating for 3h at room temperature;
(7) washing the coupled complex with PBS, and then suspending the washed coupled complex in 1m L PBS solution to obtain the CTCs magnetic nano capture probe solution.
Example 1
The enrichment and separation of the CTCs in the cell suspension comprises the following steps:
(1) HEK293 cells are cultured to 70% -80% confluence degree, and then are digested by pancreatin, blown, beaten and mixed evenly to prepare single cell suspension (Cell suspension concentration of 106/m L, simulating monocytes in blood), adding 1m L into a centrifuge tube coated with 2m L bovine serum albumin;
(2) adding pre-stained cervical cancer cells (Hela) with different numbers into HEK293 cell suspension to simulate CTCs;
(3) uniformly mixing the cell suspension, adding 100 mu L folic acid modified CTCs magnetic nano capture probe solution, and placing on a rotary mixer for continuous incubation for 15min at room temperature;
(4) then placing the centrifuge tube in a 0.6T magnetic frame, carrying out magnetic separation for 2min, and absorbing and removing the supernatant;
(5)100 μ L of PBS containing 1% BSA to resuspend the magnetic nanoprobe-CTCs complexes;
(6) spreading the heavy suspension on a glass slide, and observing and counting under a fluorescence microscope;
(7) and (3) calculating the capture efficiency: capture efficiency [ number of magnetically isolated cancer cells/labeled cancer cells ] × 100%.
The capture efficiency for each group is shown in table 1, as follows:
TABLE 1
| Number of added marks | 23 | 43 | 112 | 141 | 204 | 257 | 290 |
| Number of captures | 21 | 39 | 107 | 129 | 197 | 236 | 266 |
| Efficiency of capture | 91.3％ | 90.7％ | 87.7％ | 91.5％ | 96.6％ | 91.8％ | 91.7％ |
Experimental results show that the folic acid modified CTCs magnetic nano capture probe can efficiently enrich target tumor cells from complex background cells. The number of tumor cells in peripheral blood of cancer patients with different progressive stages is simulated by adding the tumor cells with different concentrations into the cell suspension, and the CTCs magnetic nano capture probe shows excellent and efficient targeted enrichment capacity.
Example 2
The capture of CTCs in blood, comprising the following steps:
(1) adding 1m L blood into a 10m L centrifuge tube, adding pre-dyed cervical cancer cells (Hela) into the blood, simulating CTCs, and gently blowing and stirring by a pipette to mix uniformly;
(2) adding 3m of L erythrocyte lysate into a centrifuge tube, standing the centrifuge tube for 10min, and shaking for several times;
(3) uniformly mixing the cell suspension, adding 100 mu L folic acid modified CTCs magnetic nano capture probe, placing on a rotary mixer, and continuously incubating for 15min at room temperature;
(4) after the cleavage is finished, 2000rpm is carried out for 3min, the supernatant is aspirated and discarded, 1m L PBS is used for resuspension and precipitation, and then the supernatant is transferred to a 2m L BSA coated centrifuge tube;
(5) adding 100 mu L folic acid modified CTCs magnetic nano capture probe solution, placing on a rotary mixer, and continuously incubating for 15min at room temperature;
(6) then placing the centrifuge tube in a 0.6T magnetic frame, carrying out magnetic separation for 2min, and absorbing and removing the supernatant;
(7)100 μ L of PBS containing 1% BSA to resuspend the magnetic nanoprobe-CTCs complexes;
(8) spreading the heavy suspension on a glass slide, and observing and counting under a fluorescence microscope;
(9) and (3) calculating the capture efficiency: capture efficiency [ number of magnetically isolated cancer cells/labeled cancer cells ] × 100%.
The capture efficiency is shown in table 2:
TABLE 2
Experimental results show that the magnetic nano-capture probe for CTCs can realize high-efficiency and rapid capture of CTCs in blood, and can be applied to rapid capture of CTCs in blood samples of clinical tumor patients.
Example 3
The release efficiency verification of the magnetically trapped CTCs comprises the following steps:
(1) in the embodiment, about 200 pre-stained Hela cells are added into HEK293 cell suspension of 1m L, the centrifuge tube is reversed and mixed evenly for a plurality of times, then CTCs magnetic nano capture probe solution of 100 mu L is added, and the mixture is placed above the rotary mixing and incubated at room temperature for 15min continuously;
(2) then placing the centrifuge tube in a 0.6T magnetic frame, carrying out magnetic separation for 2min, and absorbing and removing the supernatant;
(3) resuspending the magnetic nanoprobes-CTCs complex in 1m L release solution containing CTCs, and placing in a constant-temperature water bath at 37 ℃ for continuous incubation for 30 min;
(4) then placing the centrifuge tube in a 0.6T magnetic frame, carrying out magnetic separation for 2min, and absorbing and removing the supernatant;
(5)100 μ L of PBS containing 1% BSA to resuspend the magnetic nanoprobe-CTCs complexes;
(6) the heavy suspension is spread on a glass slide and placed under a fluorescence microscope for observation and counting.
(7) And (3) calculating the release efficiency: the release efficiency was [1- (number of cancer cells magnetically enriched for the second time/number of cancer cells magnetically enriched for the first time) ] 100%.
The release efficiency results are shown in table 3:
TABLE 3
| Sample number/DTT treatment time | 30min | 45min |
| 1 | 81.6％ | 77.7％ |
| 2 | 79.7％ | 83.1 |
| 3 | 76.3％ | 85％ |
| Mean value of | 79.2％ | 81.9％ |
Experimental results show that the magnetic nano can realize the capture and release of CTCs. The release efficiency of the magnetically trapped CTCs is close to 80 percent after the CTCs release solution is treated for 30min at 37 ℃.
Example 4
Verification of the activity of released CTCs comprising the steps of:
(1) in this example, Hela cells were digested with trypsin, blown into a single cell suspension, and diluted to 10 deg.C with a cell culture medium3～104/mL；
(2) Adding a CTCs magnetic nano capture probe solution with the particle size of 100 mu L into a cell suspension of 1m L, placing the solution above the solution and carrying out rotary mixing, and continuously incubating the solution at room temperature for 15 min;
(3) then placing the centrifuge tube in a 0.6T magnetic frame, carrying out magnetic separation for 2min, and absorbing and removing the supernatant;
(4) resuspending the magnetic nanoprobes-CTCs complex in CTCs release solution of 1m L, and incubating in a constant-temperature water bath at 37 ℃ for 30 min;
(5) then placing the centrifuge tube in a 0.6T magnetic frame, carrying out magnetic separation for 2min, and collecting the supernatant;
(6)1500rmp, 3min, removing supernatant by suction, and collecting cell precipitate;
(7)100 u L PBS heavy suspension cells, adding 2 u L1 mM Calcein-AM and 3 u L of 1.5mM PI (propidium iodide), mixing at 37 degrees C were incubated for 15 min;
(8) after washing twice with PBS, the resuspension solution was spread on a slide and placed under a fluorescence microscope for observation, photographing, and counting.
Live/dead cell staining results:
the experimental result shows that the released Hela cells basically show green fluorescence, and only a few cells show red fluorescence, which indicates that the released cells still have higher activity. The Image J software is used for statistical analysis, the content of the green fluorescent living cells accounts for 98.6%, and the result shows that the kit can realize the nondestructive capture and release of the CTCs.
Example 5
In vitro culture of released CTCs comprising the steps of:
(1) in this example, Hela cells were digested with trypsin, blown into a single cell suspension, and diluted to 10 deg.C with a cell culture medium3～104/mL；
(2) Adding a CTCs magnetic nano capture probe solution with the particle size of 100 mu L into a cell suspension of 1m L, placing the solution above the solution and carrying out rotary mixing, and continuously incubating the solution at room temperature for 15 min;
(3) then placing the centrifuge tube in a 0.6T magnetic frame, carrying out magnetic separation for 2min, and absorbing and removing the supernatant;
(4) resuspending the magnetic nanoprobes-CTCs complex in CTCs release solution of 1m L, and incubating in a constant-temperature water bath at 37 ℃ for 30 min;
(5) then placing the centrifuge tube in a 0.6T magnetic frame, carrying out magnetic separation for 2min, and collecting the supernatant;
(6)1500rmp, 3min, removing supernatant by suction, and collecting cell precipitate;
(7) the cells were resuspended in 1m L cell culture medium, transferred to a 6-well plate, and cultured in a constant temperature and humidity incubator.
In vitro culture results:
the experimental result shows that the cells are well adhered to the wall after being cultured for 24h, and almost no dead cells exist. Some cells have begun to divide and proliferate. Subsequently, the in vitro culture was continued for 48h and 72h to observe the growth of the cells, and the growth and proliferation of the cells were normal and were not obviously different from the morphology of the control group (without any treatment). The folic acid modified magnetic nano-capture probe for CTCs can realize the efficient nondestructive separation of CTCs, and the released CTCs have higher cell activity, can be directly cultured in vitro, and provides possibility for the research of the downstream of CTCs.
Example 6
The proteomics application of CTCs cultured in vitro comprises the following steps:
(1) taking the released tumor cells cultured in vitro for 72h, washing with cold PBS for 3 times, scraping the cells growing adherent to the wall in a culture dish by using a cell scraper, transferring to a centrifuge tube, centrifuging for 5min at 800g, and collecting cell precipitates to be placed in a 1.5m L low-adsorption centrifuge tube;
(2)0.5M L cell lysate (100mM Tris-HCl, 8M urea, 150mM NaCl, 1 × protease inhibitors cocktial, 1 × phosphate inhibitors cocktial, 1mM sodium vanadate, 1mM PMSF) was added to a 1.5M L centrifuge tube and left to lyse for 10min at 4 ℃;
(3) carrying out ultrasonic treatment on the lysate for 5s, pausing for 25s, and carrying out ultrasonic treatment for 1 min;
(4)12000g, centrifugation, 10min, collecting the supernatant in a new 1.5m L ultrafiltration centrifuge tube (10 KD);
(5) centrifuging at 3000g and 14 deg.C for 30min, and repeating for 2 times;
(6) adding TCEP to make its final concentration be 30mM, incubating at room temperature for 10min, adding Iodoacetamide (IAA) solution to make its final concentration be 50mM, and reacting at room temperature in dark place for 30 min;
(7) centrifuging at 3000g and 14 deg.C for several times, and replacing with ammonium bicarbonate solution to make urea concentration lower than 0.5M;
(8) the protein solution in the ultrafiltration tube was collected and, after measuring the protein concentration by the Bradford method, the ratio of protein: adding mass spectrum pancreatin according to the proportion of 50:1, and performing enzyme digestion overnight;
(9) adding 10% formic acid solution into the enzyme digestion solution to make the final concentration 5%, and desalting with Waters C18 desalting column;
(10) washing with 50% acetonitrile to remove peptide segment in the salt column, and drying;
(11) after the peptide fragment was dissolved in 0.1% formic acid solution, mass spectrometric detection analysis (L C-MS/MS) was carried out after the concentration was determined.
And (3) mass spectrum identification result:
proteomics can provide key protein information about CTCs cell heterogeneity, disease stage, and chances of survival. The non-invasive isolation and release of CTCs opens the possibility for the proteomics of CTCs. Based on the enrichment and release method of the kit, proteomics identification is carried out on model CTCs cultured in vitro after release in research, and 3754 proteins are identified in experiments.
Example 7
The detection of CTCs in blood comprises the following steps:
(1) adding 1m L blood into a 10m L centrifuge tube, adding pre-dyed cervical cancer cells (Hela) into the blood, simulating CTCs, and gently blowing and stirring by a pipette to mix uniformly;
(2) adding 3m of L erythrocyte lysate into a centrifuge tube, standing the centrifuge tube for 10min, and shaking for several times;
(3) uniformly mixing the cell suspension, adding 100 mu L folic acid modified CTCs magnetic nano capture probe, placing on a rotary mixer, and continuously incubating for 15min at room temperature;
(4) after the cleavage is finished, 2000rpm is carried out for 3min, the supernatant is aspirated and discarded, 1m L PBS is used for resuspension and precipitation, and then the supernatant is transferred to a 2m L BSA coated centrifuge tube;
(5) adding 100 mu L folic acid modified CTCs magnetic nano capture probe, placing on a rotary mixer, and continuously incubating for 15min at room temperature;
(4) then placing the centrifuge tube in a 0.6T magnetic frame, carrying out magnetic separation for 2min, and absorbing and removing the supernatant;
(5) resuspending the magnetic nanoprobes-CTCs complex in 1m L CTCs release solution, and placing in a constant-temperature water bath at 37 ℃ for incubation for 30 min;
(6) then placing the centrifuge tube in a 0.6T magnetic frame, carrying out magnetic separation for 2min, and absorbing and removing the supernatant;
(7) resuspending 200 mu L of 4% paraformaldehyde solution, smearing on a confocal culture dish, and fixing at room temperature for 30 min;
(8) after washing with PBS three times, blocking with 5% BSA at room temperature for 1 h;
(9) washing with PBS for 3 times, adding detection solution B of 1m L, and standing and incubating for 1h at room temperature;
(10) washing with PBS for 3 times, adding detection solution C1 m L, and standing at room temperature for 30 min;
(11) after washing with PBS 3 times, the cell staining results were observed under a laser confocal microscope.
The cell staining result shows that the tumor cells show red fluorescence, the cell nucleus shows blue color, and the cell size is larger than 8 μm, which is obviously different from blood cells. The result shows that the kit can be used for counting the 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 chains2K-NHS) with an MNs @ Cys conjugate complex to form MNs @ Cys @ PEG2k-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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