CN108690832B - Method for capturing and releasing circulating tumor cells - Google Patents
Method for capturing and releasing circulating tumor cells Download PDFInfo
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- CN108690832B CN108690832B CN201710236512.1A CN201710236512A CN108690832B CN 108690832 B CN108690832 B CN 108690832B CN 201710236512 A CN201710236512 A CN 201710236512A CN 108690832 B CN108690832 B CN 108690832B
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a capture and release method of circulating tumor cells, which comprises the steps of modifying enzyme-cutting tumor specific binding polypeptide into a microfluidic chip flow channel; introducing a blood sample containing tumor cells into the microfluidic chip flow channel to capture the tumor cells; and introducing selected enzyme into the microfluidic chip flow channel to cut the enzyme-cleavable tumor specific binding polypeptide, thereby realizing the release of the captured cells. The invention takes the PDMS microfluidic chip as a base, realizes capture and release of circulating tumor cells by modifying tumor cell targeting polypeptide which can be cut by specific enzyme in a flow channel, has low cost of enzyme-cutting tumor specificity combined polypeptide compared with the antibody capture of circulating tumor cells, can complete release after capture, is convenient for re-culture and further research of cells in later period, and is suitable for production and application.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a method for capturing and releasing circulating tumor cells in the field of circulating tumor cell detection.
Background
Tumors are one of the major threats facing the world, an effective means for thoroughly curing the tumors still does not exist at present, and the effective treatment of the tumors can be realized only by early detection and early diagnosis. The detection of Circulating Tumor Cells (CTCs) is effectively applied to the early diagnosis of metastatic tumors and the evaluation of therapeutic effects, but CTCs are present in very small amounts in the peripheral blood, and there are about 1 to 10 circulating tumor cells per 10ml in the peripheral blood of cancer patients. Among the currently available methods for detecting circulating tumor cells, most simple and sensitive detection methods rely on the identification of EpCAM on the surface of circulating tumor cells, which are all tumor cells expressed by EpCAM. However, the EpCAM-dependent detection method for epithelial-like CTCs has certain limitations. Firstly, in the process of tumor development and metastasis, Epithelial Mesenchymal Transition (EMT) occurs in part of cells, and the part of EMT-occurring CTCs cannot be effectively detected by an EpCAM-dependent capture method, so that omission of CTCs is caused, and a certain degree of false negative is caused; second, EpCAM antibodies are expensive to use, limiting their production and use; thirdly, since the capture of antibodies can only detect the presence of CTCs and intensive research and examination of CTCs in a minute flow channel cannot be performed, how to release CTCs from the flow channel is also an important issue.
Currently, there have been related studies to achieve capture of circulating tumor cells by antibodies or aptamers against unique antigens on the surface of circulating tumor cells. However, whether the capture of circulating tumor cells can be efficiently accomplished by other biomolecules is a problem to be explored by finding a relatively inexpensive method for capturing and obtaining circulating tumor cells without damage.
Disclosure of Invention
In view of the defects in the prior art, the main object of the present invention is to provide a method for capturing and releasing circulating tumor cells in a microfluidic chip by using an enzyme-cleavable tumor-specific binding polypeptide, wherein the method has relatively low cost and can simultaneously capture and release the circulating tumor cells.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
a method for capturing and releasing circulating tumor cells comprises the following steps:
(1) modifying the enzyme-cleavable tumor specific binding polypeptide into a microfluidic chip flow channel;
(2) introducing a blood sample containing tumor cells into the microfluidic chip flow channel to capture the tumor cells;
(3) and introducing selected enzyme into the microfluidic chip flow channel to cut the enzyme-cleavable tumor specific binding polypeptide, thereby realizing the release of the captured cells.
In one embodiment, the modification method in step (1) includes a modification by utilizing an affinity interaction between the modified avidin in the flow channel of the microfluidic chip and the biotin labeled at one end of the enzyme-cleavable tumor-specific binding polypeptide, and/or a chemical modification method.
In one embodiment, the chemical modification method is thiol modification.
In one embodiment, the enzymatically cleavable tumor-specific binding polypeptide has the sequence WFCSWYGGDTCVQGGENLYFQGGGK-Biotin; wherein, the targeting sequence is as follows: WFCSWYGGDTCVQ, the enzyme cutting sequence is: ENLYFQ.
In one embodiment, step (2) comprises: the specific binding effect of the enzyme-cleavable tumor specific binding polypeptide on tumor cells is utilized to realize the capture of specific tumor cells.
In one embodiment, before step (2), the method further comprises: and (2) introducing a tumor cell suspension liquid with a certain concentration into the microfluidic chip flow channel modified in the step (1) to verify the function of capturing the circulating tumor cells.
In one embodiment, step (2) is preceded by the step of pre-treating the blood sample by: subjecting a blood sample containing tumor cells to red blood cell lysis such that circulating tumor cells in the blood sample are enriched to a predetermined concentration.
In one embodiment, subjecting a blood sample containing tumor cells to red blood cell lysis such that circulating tumor cells in the blood sample are enriched to a predetermined concentration specifically comprises: adding erythrocyte lysate into blood sample containing tumor cells, repeating the steps of centrifuging at least once, removing supernatant, and adding PBS for resuspension and precipitation.
Compared with the prior art, the invention has the beneficial effects that:
(1) compared with the traditional antibody, the polypeptide not only can be specifically combined with tumor cells to realize capture, but also can realize lossless release of the captured cells by adding an enzyme-cutting sequence through design, has low cost and creates possibility for deeper research.
(2) The targeted polypeptide has diversity for capturing tumor cells, is not limited to the enzyme-cleavable A-1 targeted polypeptide described in the invention, and the enzyme-cleavable site can also be designed in various ways, and by utilizing other targeted polypeptides and other enzyme-cleavable sites, a microfluidic chip with higher capture efficiency and sensitivity can be further designed, so that the research space is wider, and the prospect is wider.
(3) The polypeptide-modified microfluidic chip is easy to prepare and low in cost.
Drawings
FIG. 1 is a schematic diagram of the capturing and releasing process of tumor cells in a microfluidic chip flow channel modified by an enzyme-cleavable tumor-specific binding polypeptide according to an embodiment of the capturing and releasing method of circulating tumor cells of the present invention;
FIG. 2 is a schematic diagram showing the capturing ability of the microfluidic chip modified by the enzyme-cleavable tumor-specific binding polypeptide to tumor cells according to an embodiment of the method for capturing and releasing circulating tumor cells of the present invention;
FIG. 3 is a schematic diagram showing the cell releasing ability of the enzyme in the microfluidic chip modified by the enzyme-cleavable tumor-specific binding polypeptide according to an embodiment of the method for capturing and releasing circulating tumor cells of the present invention.
Detailed Description
The invention is described in more detail below with reference to examples and figures, but the scope of the invention is not limited thereto.
Referring to fig. 1, an embodiment of the method for capturing and releasing circulating tumor cells of the present invention is described, which comprises the following steps:
step S1: and (4) preparing the microfluidic chip.
In one embodiment, the preparation method of the microfluidic chip specifically comprises the steps of mixing a PDMS precursor and a curing agent 10:1, uniformly stirring, pouring the mixture on a silicon wafer mold with a runner pattern, controlling the thickness to be 5mm, and placing the silicon wafer mold in a vacuum drier for vacuumizing to remove bubbles. Curing at 90 ℃ for 1 h. The PDMS block was cut out with a cutter and holes were made at the exit and entrance of the flow channel. Putting the cleaned glass slide and PDMS block into a PLASMA Cleaner, vacuumizing until the air pressure in the apparatus is 500mTorr, opening the PLASMA action for 30s, taking out, attaching the PDMS block to the glass slide, lightly pressing, and baking in a drying oven at 90 ℃ for 5 minutes to make the PDMS block and the glass slide permanently bonded.
Step S2: modifying the enzyme-cleavable tumor specific binding polypeptide into a microfluidic chip flow channel.
The modification method mentioned in this step includes modification by utilizing an affinity interaction between avidin modified in a flow channel of the microfluidic chip and biotin labeled at one end of the enzyme-cleavable tumor specific binding polypeptide, and/or a chemical modification method, and further exemplarily, the chemical modification method is thiol modification.
In one embodiment, the method for modifying avidin in a flow channel specifically comprises: and introducing an ethanol solution of 2% (v/v) silane reagent at the flow rate of 10 mu L/min under the condition of room temperature and light shielding, introducing the ethanol solution at the flow rate of 10 mu L/min, cleaning for 20 minutes, introducing deionized water at the flow rate of 10 mu L/min, cleaning for 30 minutes, blow-drying the liquid in the chip, and baking at the temperature of 110 ℃ for 60 minutes. After the chip is cooled, glutaraldehyde reaction liquid is introduced for 1 hour at the flow rate of 10 mu L/min (the preparation of the glutaraldehyde reaction liquid is 1mL of 50% glutaraldehyde, PBS20mL, sodium cyanoborohydride 40mg, and the pH value is 7.4), deionized water is introduced at the flow rate of 10 mu L/min for washing for 30 minutes, the liquid in the chip is dried, and then 100 mu g/mL of avidin PBS solution is introduced and stored at the temperature of 4 ℃. Wherein the ethanol solution of the silane reagent can be selected from 3-aminopropyltriethoxysilane.
In one embodiment, the enzyme-cleavable tumor-specific binding polypeptide modification (labeled biotin) method specifically comprises: and (2) introducing deionized water into a flow channel of the microfluidic chip at the flow rate of 10 mu L/min for washing for 10 minutes to remove unconjugated avidin, introducing 1mg/mL of a PBS (phosphate buffer solution) solution of biotinylated and enzyme-cleavable tumor specific binding polypeptide into the micro flow channel, incubating for 45 minutes, introducing the PBS at the flow rate of 10 mu L/min for washing for 10 minutes, introducing a 2% (w/v) BSA (bovine serum albumin) solution for sealing for 1 hour, introducing the PBS at the flow rate of 10 mu L/min for washing for 10 minutes, and placing at 4 ℃ for later use.
In one embodiment, the enzyme-cleavable tumor-specific binding polypeptide has the sequence WFCSWYGGDTCVQGGENLYFQGGGK-Biotin; wherein, the targeting sequence is as follows: WFCSWYGGDTCVQ, the enzyme cutting sequence is: ENLYFQ, the remaining sequence being the linker sequence.
Step S3: and (4) introducing a tumor cell suspension with a certain concentration into the flow channel of the microfluidic chip modified in the step S1 to verify the function of capturing the circulating tumor cells.
Step S4: pre-treating a blood sample: subjecting a blood sample containing tumor cells to red blood cell lysis such that circulating tumor cells in the blood sample are enriched to a predetermined concentration.
The step may specifically include the steps of adding a red blood cell lysate to a blood sample containing tumor cells, repeating the centrifugation at least once, removing the supernatant, and adding PBS to resuspend the pellet. In one example, 1mL of fresh anticoagulation blood (containing tumor cells A549) is added with 10mL of erythrocyte lysate (purchased from TBDscience, with the product number of NH4CL2009) and gently blown and uniformly mixed, and the mixture is lysed for 2 minutes at room temperature; then centrifuged at 500g4 ℃ for 5 minutes, and the red supernatant was discarded; and adding 5mL of PBS for resuspending the precipitate, centrifuging at 500g and 4 ℃ for 3 minutes, removing the supernatant, repeating for 1 time, washing for 2 times totally, and resuspending the cell precipitate in 1mL of PBS to realize the enrichment of the circulating tumor cells.
Step S5: and introducing the enriched blood sample containing the tumor cells into the microfluidic chip flow channel to capture the tumor cells.
The specific binding effect of the enzyme-cleavable tumor specific binding polypeptide on tumor cells can be utilized to realize the capture of specific tumor cells. For example, in this embodiment, the enzymatically cleavable tumor-specific binding polypeptide is capable of specifically binding to human non-small cell lung cancer cell a 549. Pancreatin digests A549 cells, PBS dilutes the cells into cell suspension, then the cell suspension is introduced into the flow channel of the microfluidic chip at the flow rate of 1 mu L/min, and the cells in the flow channel are counted under a microscope, so that each flow channel contains 100 cells and 200 cells; finally, the cells that were not captured were washed off by washing with PBS at a flow rate of 1. mu.L/min for 5 min.
As shown in fig. 2, another polypeptide not binding to a549 was used as a control, and there were few tumor cells remained in the flow channel modified by the control polypeptide after PBS washing, whereas the enzymatically cleavable tumor-specific binding polypeptide in this embodiment has a better capturing effect on a 549.
Step S6: and introducing selected enzyme into the microfluidic chip flow channel to cut the enzyme-cleavable tumor specific binding polypeptide, thereby realizing the release of the captured cells.
In one example, the captured cells were released by applying an enzyme solution (PBS on the control chip, otherwise the conditions were consistent), allowing the cells to act at room temperature for 15min, and then washing the cells with PBS at a flow rate of 1 μ L/min for 5 min. The remaining cells in the flow channel were counted under a microscope.
As shown in FIG. 3, only a small number of cells were washed down due to the binding of the polypeptide by introducing PBS after capturing tumor cells in the control chip, whereas most of the tumor cells were released after introducing the enzyme solution in the experimental chip.
It should be noted that the tumor cells and the enzymatically cleavable tumor-specific binding polypeptides mentioned in the above embodiments correspond to each other, for example, the tumor cell corresponding to the enzymatically cleavable tumor-specific binding polypeptide in this embodiment is a549, while in other embodiments, the tumor cell and the targeting polypeptide may be replaced by any combination capable of specifically binding to each other, and the enzyme cleavage sequence may be any other sequences capable of being cleaved.
In conclusion, the invention is based on the PDMS microfluidic chip, realizes capture and release of the circulating tumor cells by modifying the tumor cell targeting polypeptide which can be cut by specific enzyme in the flow channel, has low cost of the enzyme-cutting tumor specific binding polypeptide compared with the antibody capture of the circulating tumor cells, can complete the release after capture, is convenient for the later re-culture and further research of the cells, and has wide application prospect in the field of detection of the circulating tumor cells.
It should be noted that the above-mentioned embodiments of the present invention do not limit the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Claims (4)
1. A method for capturing and releasing circulating tumor cells for non-medical purposes, comprising the steps of:
(1) modifying the enzyme-cleavable tumor specific binding polypeptide into the microfluidic chip flow channel by utilizing the affinity effect between the modified avidin in the microfluidic chip flow channel and biotin marked at one end of the enzyme-cleavable tumor specific binding polypeptide;
(2) introducing a blood sample containing tumor cells into the microfluidic chip flow channel, and capturing specific tumor cells by utilizing the specific binding effect of the enzyme-cleavable tumor specific binding polypeptide on the tumor cells;
(3) introducing selected enzyme into the microfluidic chip flow channel to cut the enzyme-cleavable tumor specific binding polypeptide, thereby realizing the release of the captured cells;
the sequence of the enzyme-cleavable tumor specific binding polypeptide is WFCSWYGGDTCVQGGENLYFQGGGK-Biotin, wherein the targeting sequence is: WFCSWYGGDTCVQ, the enzyme cutting sequence is: ENLYFQ.
2. The method of claim 1, further comprising, prior to step (2): and (2) introducing a tumor cell suspension liquid with a certain concentration into the microfluidic chip flow channel modified in the step (1) to verify the function of capturing the circulating tumor cells.
3. The method for capturing and releasing circulating tumor cells of claim 1, wherein the step (2) is preceded by the step of pre-treating the blood sample by: subjecting a blood sample containing tumor cells to red blood cell lysis such that circulating tumor cells in the blood sample are enriched to a predetermined concentration.
4. The method of claim 3, wherein the step of lysing the blood sample containing the tumor cells to enrich the blood sample in circulating tumor cells to a predetermined concentration comprises: adding erythrocyte lysate into blood sample containing tumor cells, repeating the steps of centrifuging at least once, removing supernatant, and adding PBS for resuspension and precipitation.
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| CN110643482A (en) * | 2019-08-27 | 2020-01-03 | 宁波美晶医疗技术有限公司 | Preparation method and application of nano-structure surface chip for capturing and releasing circulating tumor cells |
| CN112924363A (en) * | 2021-01-22 | 2021-06-08 | 中国科学院苏州纳米技术与纳米仿生研究所 | Intermediate circulating tumor cell as tumor diagnosis and prognosis marker and application thereof |
| CN113713866A (en) * | 2021-08-05 | 2021-11-30 | 武汉大学 | Human bone microfluidic chip embedded in cancer cell membrane, preparation method thereof and application of chip in separation of circulating tumor cells |
| CN114249834B (en) * | 2021-12-23 | 2023-06-23 | 中国科学院苏州纳米技术与纳米仿生研究所 | Chimeric antigen receptor capable of specifically targeting tumor cells, expressed gene thereof, modified NK cells and application thereof |
| CN114712521A (en) * | 2022-03-22 | 2022-07-08 | 郑州大学 | CD44 receptor-targeted drug, and preparation method and application thereof |
| CN114874989A (en) * | 2022-04-11 | 2022-08-09 | 中国科学院苏州纳米技术与纳米仿生研究所 | Method for capturing circulating tumor cells |
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