Methods, kits, and applications for detecting epithelial-mesenchymal transition in cells.

Abstract

This invention provides a method, kit, and application for detecting epithelial-mesenchymal transition (EMT) in cells. The method includes: 1) incubating an integrin antibody carrying a single biotin with cells to be tested to specifically recognize and bind to integrins on the cell membrane surface of the cells; after incubation, removing unbound integrin antibodies carrying a single biotin; 2) adding a fluorescent probe carrying streptavidin to the product obtained in step 1) and incubating; after the reaction, removing unreacted excess fluorescent probe carrying streptavidin to obtain fluorescently labeled integrins; 3) imaging the fluorescent probe of the fluorescently labeled integrins obtained in step 2) and detecting the change in the average diffusion rate of the fluorescently labeled integrins relative to control cells, wherein an increase in the average diffusion rate of the fluorescently labeled integrins relative to control cells indicates that epithelial-mesenchymal transition has occurred. This invention offers higher sensitivity and accuracy, effectively reducing the false negative rate in measurements.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for detecting epithelial-mesenchymal transition in cells. Background Technology

[0002] Epithelial-mesenchymal transition (EMT) refers to the transformation of epithelial cells into mesenchymal cells, a crucial biological process significant for studying cell differentiation, embryonic development, cancer metastasis, and tissue healing. EMT results in cells acquiring strong invasive capabilities and increased cell migration. Common markers of EMT include: 1. Changes in cell morphology from fish-scale to spindle-shaped; 2. Decreased levels of E-cadherin, a key cell-cell junction marker; 3. Changes in the cytoskeleton, such as the appearance of prominent plate-like pseudopodia; and 4. Abundant intermediate filaments. Therefore, common methods for measuring EMT primarily involve measuring changes in cell morphology or E-cadherin levels. However, neither of these methods currently provides a perfect distinction between cells induced by EMT.

[0003] During cell migration, focal adhesions (FAS), the connections between cells and the extracellular matrix (ECM), play a crucial role. Filomerae are dynamic molecular aggregates that continuously break down and reassemble. Integrins are important components of FAS, and their formation can be controlled by regulating the affinity of integrins for the ECM. Integrins are transmembrane proteins that exist in both activated and inactivated states, switching between them. When inactive, they move along the cell membrane. When activated, they recruit proteins such as Talin to connect the ECM to actin, forming contractile units. After activation, one end of the integrin is connected to the external environment, and the other end is connected to actin, resulting in a temporarily fixed state. The complex formed by multiple integrins matures to form a focal adhesion (FAS). During cell migration, this physical connection at the focal adhesion site pulls the cell forward. Therefore, integrins are essential proteins closely related to cell migration.

[0004] Current research has extensively investigated integrin labeling and motility, including different motility states of integrins inside and outside focal adhesion sites, integrin arrest and activation, and integrin diffusion behavior. Most current single-molecule studies on integrins focus on the formation of focal adhesion sclerosing agents (FAS) and integrin activation. Additionally, some studies, as in vivo continuations of in vitro single-molecule integrin research, address how integrins participate in focal adhesion behavior at a small scale, rather than focusing on large-scale issues such as cell crawling. Currently, research on integrins and EMT is limited to studies related to biological signaling pathways, and there are no studies combining integrin dynamics with EMT. Summary of the Invention

[0005] One object of this invention is to provide a novel method for detecting epithelial-mesenchymal transition (EMT) in cells. Unlike previous methods that labeled integrins by binding to ligand-quantum dot (QD) / GFP, the inventors unexpectedly discovered that labeling integrins using a primary antibody carrying a single biotin and a fluorescent probe carrying streptavidin allows for the differentiation between E-type and M-type cells through integrin diffusion, thereby enabling the detection of EMT. Based on this discovery, this invention was completed.

[0006] Another object of the present invention is to provide a kit for detecting epithelial-mesenchymal transition in cells.

[0007] Another object of the present invention is to provide the use of the kit.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] On one hand, the present invention provides a method for detecting epithelial-mesenchymal transition in cells, the method comprising the following steps:

[0010] 1) Incubate the cells to be tested with an integrin antibody carrying a single biotin to specifically recognize and bind to integrins on the cell membrane surface of the cells. After incubation, remove the unbound integrin antibody carrying a single biotin.

[0011] 2) Add a fluorescent probe containing streptavidin to the product obtained in step 1) and incubate. After the reaction, remove the unreacted excess fluorescent probe containing streptavidin to obtain fluorescent probe-labeled integrin.

[0012] 3) By imaging the fluorescent probe of the integrin labeled with the fluorescent probe obtained in step 2), the change in the average diffusion rate of the fluorescent probe-labeled integrin relative to the control cells is detected, wherein the increase in the average diffusion rate of the fluorescent probe-labeled integrin relative to the control cells indicates that the cells have undergone epithelial-mesenchymal transition.

[0013] Preferably, the average diffusion rate of the integrin labeled by the fluorescent probe is increased by 40%-172% compared to control cells; more preferably, the average diffusion rate of the integrin labeled by the fluorescent probe is increased by 80%-172% compared to control cells.

[0014] Preferably, a cell pretreatment step is included before step 1).

[0015] Preferably, the cells are selected from one or more of epithelial cells, mesenchymal cells, tumor cells, peripheral blood mononuclear cells or lymphocytes; more preferably, the cells are epithelial cells or mesenchymal cells; most preferably, the cells are normal breast cells MCF10A or breast cells MCF10A after epithelial-mesenchymal transition.

[0016] Preferably, the control cells are selected from cells that have not undergone epithelial-mesenchymal transition; more preferably, the control cells are epithelial cells; most preferably, the control cells are normal breast cells MCF10A.

[0017] Preferably, in step 1), the incubation time is 10 to 18 minutes and the incubation temperature is 0 to 4°C; more preferably, the incubation time is 15 minutes and the incubation temperature is 0°C.

[0018] Preferably, the integrin antibody containing a single biotin is selected from one or more of the following: anti-β1 integrin antibody containing a single biotin, anti-α5 integrin antibody containing a single biotin, anti-β3 integrin antibody containing a single biotin, and anti-αv integrin antibody containing a single biotin; more preferably, the integrin antibody containing a single biotin is an anti-β1 integrin antibody or an anti-α5 integrin antibody containing a single biotin; most preferably, the integrin antibody containing a single biotin is an anti-β1 integrin antibody containing a single biotin.

[0019] Preferably, in step 2), the incubation time is 3 to 8 minutes and the incubation temperature is 0 to 4°C; more preferably, the incubation time is 5 minutes and the incubation temperature is 0°C.

[0020] Preferably, the fluorescent probe is selected from one or more of fluorescent proteins, polymer dyes, or quantum dots; more preferably, the fluorescent probe is a quantum dot; even more preferably, the quantum dot is selected from CdTe, CdSe, and CdSe / ZnS core-shell quantum dots, preferably CdSe / ZnS core-shell quantum dots.

[0021] Preferably, the quantum dot is selected from one or more quantum dots with a maximum emission wavelength of 525nm-800nm; more preferably, the quantum dot is a quantum dot with a maximum emission wavelength of 600nm-800nm; most preferably, the quantum dot is a quantum dot with a maximum emission wavelength of 655nm.

[0022] Preferably, in step 3), the emission wavelength of the imaging is 525nm-800nm; more preferably, the emission wavelength of the imaging is 600nm-800nm; most preferably, the emission wavelength of the imaging is 655nm.

[0023] Preferably, the excitation wavelength of the imaging is smaller than the emission wavelength of the imaging.

[0024] Preferably, before step 2), a step of centrifuging the fluorescent probe containing streptavidin is included.

[0025] Preferably, the centrifugation is performed at 3000-7000g for 2-5 minutes; more preferably, the centrifugation is performed at 5000g for 3-4 minutes.

[0026] On the other hand, the present invention provides the use of integrin antibodies with a single biotin and fluorescent probes with streptavidin in the preparation of kits for detecting epithelial-mesenchymal transition in cells.

[0027] In another aspect, the present invention provides a kit for detecting epithelial-mesenchymal transition in cells, the kit comprising: an integrin antibody with a single biotin and a fluorescent probe with streptavidin.

[0028] Preferably, the integrin antibody containing a single biotin is selected from one or more of the following: anti-β1 integrin antibody containing a single biotin, anti-α5 integrin antibody containing a single biotin, anti-β3 integrin antibody containing a single biotin, and anti-αv integrin antibody containing a single biotin; preferably, the integrin antibody containing a single biotin is an anti-β1 integrin antibody or an anti-α5 integrin antibody containing a single biotin; more preferably, the integrin antibody containing a single biotin is an anti-β1 integrin antibody containing a single biotin.

[0029] Preferably, the fluorescent probe is selected from one or more of fluorescent proteins, polymer dyes, or quantum dots; more preferably, the fluorescent probe is a quantum dot; even more preferably, the quantum dot is selected from CdTe, CdSe, and CdSe / ZnS core-shell quantum dots, preferably CdSe / ZnS core-shell quantum dots.

[0030] Preferably, the quantum dots are selected from one or more quantum dots with a maximum emission wavelength of 525nm-800nm; more preferably, the quantum dots are quantum dots with a maximum emission wavelength of 600nm-800nm; most preferably, the quantum dots are quantum dots with a maximum emission wavelength of 655nm. This invention is the first to combine and study integrin dynamics with EMT, clarifying the relationship between integrin diffusion and cell migration ability. This has significant implications for EMT-related research and can be extended to other cell research fields, such as the study of the relationship between integrin dynamics and cell morphology or cell adhesion. Compared with the prior art, this invention has at least the following advantages:

[0031] 1. The method of the present invention improves upon the prior art of labeling integrins with primary and secondary antibodies by employing quantum dots with stable and durable brightness, thereby making the detection system more stable and maintaining stable and excellent brightness.

[0032] 2. Compared with the primary and secondary antibody labeling of the prior art, the method of the present invention can ensure a 1:1 relationship between the labeled integrin and the quantum dot, and avoid the situation where one primary antibody is connected to multiple secondary antibodies.

[0033] 3. The method of the present invention has a short cycle time and is faster than commonly used fluorescence expression methods, which can meet the needs of rapid determination.

[0034] 4. The method of the present invention is applicable to various cells with different invasive abilities and different mobility.

[0035] 5. The method of the present invention is carried out in living cells. Compared with the traditional method, it does not change the genes inside the cell, does not require killing the cell for fixation, and can still retain the function of integrin after labeling. The labeled integrin can participate in the formation of adhesion clusters, with little impact on the cell and more accurate results.

[0036] 6. Compared with methods for measuring cell morphology changes, the method of the present invention has higher sensitivity and accuracy, and can effectively reduce the false negative rate in the measurement.

[0037] 7. Compared with the method for measuring calcarein, the method of the present invention takes less time and has simpler experimental steps.

[0038] 8. The method of the present invention can be applied to labeling various integrins and has guiding significance for the study of integrin diffusion in various types of cells. Attached Figure Description

[0039] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0040] Figure 1 A shows that the morphology of MCF10A cells gradually changed after EMT.

[0041] Figure 1 B shows the statistical changes in cell roundness and area before and after EMT.

[0042] Figure 2 A shows a fluorescence image of integrins on the surface of labeled cells, captured by a fluorescence microscope.

[0043] Figure 2 B shows the diffusion trajectory of a single integrin obtained after 2000 consecutive frames (0.1s interval).

[0044] Figure 3A shows the mean MSD diffusion curves of integrins on E-type and M-type cells, as well as the mean MSD diffusion curves of integrins on MCF7 and MDA-MB-231 cells.

[0045] Figure 3 B shows the diffusion rate of integrin on MCF10A, MCF7, and MDA-MB-231 cells before and after EMT induction, obtained from the MSD diffusion curve.

[0046] Figure 4 This diagram shows the correlation between the diffusion rate of integrins on cells and cell roundness, as well as the distribution of each.

[0047] Figure 5 An image showing quantum dot-tagged α5 integrin.

[0048] Figure 6 A shows fluorescence imaging of quantum dot-labeled integrins and talin proteins within adhesion spots using a dual-channel imaging system, obtaining images of their fluorescent binding.

[0049] Figure 6 B shows the relative light intensity of newly formed talin protein at the edge compared to the quantum dots.

[0050] Figure 7 This shows the proportion of cell fixation trajectories of integrins after Mn-activated quantum dot labeling.

[0051] Figure 8 The image shows a comparison of images after integrin is labeled with a primary antibody and a secondary antibody containing fluorescent protein, and after integrin is labeled using the method of the present invention.

[0052] Figure 9 This image shows an image of cadherin used in the study of epithelial-mesenchymal transition (EMT) via cadherin assays. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0054] Example 1 EMT induction in cells

[0055] EMT induction was performed on non-carcinogenic MCF10A cells by adding TGF-β1 using a common method and incubating for more than 72 hours in a cell culture incubator at 37°C and 5% CO2. Figure 1 As can be seen, the cell morphology changes after EMT induction. Figure 1In B, the area and roundness of cells (cell count > 30) were analyzed, and it was found that the average spreading area increased while the average roundness decreased. However, the ranges of roundness and spreading area overlapped to varying degrees, making it impossible to directly distinguish whether cells had undergone EMT.

[0056] The roundness is calculated using the following formula; the closer the value is to 1, the closer the cell is to a perfect circle:

[0057] Roundness = 4π * area / perimeter 2 .

[0058] Example 2 Dynamics of quantum dot-tagged β1 integrin

[0059] We employed quantum dots for β1-integrin labeling. The labeling experiment was performed on ice to pause cellular physiological activity. Cells were first incubated on ice (approximately 0°C) for 15 min with a monoclonal antibody, Anti-ITGB1 (MEM-101), containing a single biotin, to specifically bind β1-integrin to the cell membrane. Cells were then washed three times with PBS. Streptavidin-QD (655 nm, Invitrogen) was centrifuged at 50 rpm for 3 min, and the supernatant was aspirated and added to the cells that had been bound to the primary antibody. Cells were incubated on ice (approximately 0°C) for 5 min to allow binding. After washing away excess QD (three times with PBS), cells were incubated in a culture chamber (37°C, 5% CO2) for 15 min before fluorescence imaging. Glass culture dishes were placed at a 60x oil immersion depth and photographed using an EMCCD camera. Excited by a 561nm laser, the emitted light signal at 655nm was captured to obtain multiple motion videos of QD-labeled β1 integrins (shooting interval 10Hz, 2000 frames per shot, shooting time 30-50 minutes). The motion of the β1 integrins was tracked using the Particle Tracker plugin in ImageJ software, and diffusion trajectories longer than 30 frames (3 seconds) were selected for analysis.

[0060] The results are as follows Figure 2 and Figure 3 As shown. Figure 2 A shows the fluorescent labeling pattern of the quantum dots we obtained on the surface of a single cell. Figure 2 B shows the diffusion trajectory of quantum dot-β1 integrin 1 obtained after continuous imaging. The motion information of these trajectories over time was extracted and analyzed. From the mean square displacement (MSD) curve of the integrin diffusion trajectory and the diffusion rate, the diffusion rate of integrin in M-type cells is significantly faster than that in E-type cells. Figure 3A shows that the MSD curve for integrin diffusion on M-type cells is higher than that on E-type cells, and this difference is consistent with the difference between the weakly metastatic cancer cell MCF7 and the strongly metastatic cancer cell MDA-MB-231 that we used to validate this. Figure 3 B shows the diffusion velocity calculated from the MSD curve. It provides a more visual representation of the difference in integrin movement between type E (epithelial) cells and type M (mesenchymal) cells. The average diffusion velocity ranges from 0.037 μm. 2 / s increased to 0.067μm 2 / s, with an increase of approximately 80%, while the median increased from 0.03μm. 2 / s increased to 0.06μm 2 / s, an increase of 100%. Specifically, the average maximum diffusion rate of M-type cells increased by 172% compared to the average minimum diffusion rate of E-type cells. This indicates a significant increase in integrin diffusion after EMT induction. Integrin kinetics differ between E-type and M-type cells. From Figure 4 The distribution maps showing cell roundness and integrin diffusion rate indicate that the increased integrin diffusion rate induced by EMT is independent of cell morphology. Whether in cells with low roundness and pronounced morphological polarity, or in surrounded cells with high roundness and less pronounced morphological polarity, M-type cells exhibit a higher average diffusion rate than E-type cells. Therefore, this can serve as a new and more reliable way to differentiate cells before and after EMT induction.

[0061] Example 3 Dynamics of quantum dot-tagged α5 integrin

[0062] The experimental conditions were the same as in Example 2, except that the monoclonal antibody carrying a single biotin was replaced with an anti-α5 integrin antibody. Results are shown below. Figure 5 It can be seen that, in terms of labeling effect, α5 integrins labeled with quantum dots are not as numerous as those labeled with β1 integrins. This is because α5 can only have one integrin, α5β1, but the diffusion trajectory of integrins can still be detected very well.

[0063] Example 4 Dual-channel imaging of integrin and talin proteins

[0064] Simultaneous fluorescence imaging was performed on β1 integrin and talin protein within the focal adhesion. First, GFP-talin was added to the cell culture medium in culture dishes and incubated overnight (12-18 h). Then, integrin was labeled with quantum dots as described in Example 2, and both were imaged simultaneously. GFP-talin was excited with a 488 nm laser, and the quantum dots were excited with a 561 nm laser, yielding fluorescence images from two channels, which were then combined (see...). Figure 6Cell movement was observed through long-term (2h) fluorescence imaging video, and the labeled integrins were seen to reach the leading edge of the cell and appear together with the newly formed talin protein at the leading edge. This indicates that they can participate in the formation of adhesion focals at the leading edge of the cell. This shows that the labeled integrins can still perform the function of integrins, that is, co-localize with talin protein and participate in adhesion focals.

[0065] Example 5 Verification Experiment 1

[0066] Validation was performed using low-metastatic MCF7 cancer cells and high-metastatic MDA-MB-231 cancer cells. First, as described in Example 2, integrins on both cell types were labeled with quantum dots. Then, fluorescence imaging was performed and analyzed, and the MSD values ​​were calculated by extracting the integrin diffusion trajectories. Results are as follows: Figure 3 As shown in Figures A and B, the MSD curve for integrin diffusion on MCF7 cells is lower than that on MDA-MB-231 cells, indicating a lower diffusion rate. This suggests that the motility of integrins on MCF7 cells is weaker than that on MDA-MB-231 cells. This experiment verifies that the changes in integrin motility on the EMT are real, indicating that when cells acquire high invasiveness or strong motility, the diffusion and motility of integrins on the cell membrane also increase. Therefore, there is a positive correlation between integrin diffusion and cell motility.

[0067] Example 6 Verification Experiment 2

[0068] Mn ions are known to activate integrins, placing them in their active conformation, at which point the integrins are immobilized. Therefore, Mn can be used to verify the function of labeled integrins. During fluorescence microscopy imaging, MCF10A cells were first photographed normally, then 0.1 mM MnCl2 was added, and after 10 minutes, another photograph was taken. The proportion of immobilized integrin trajectories on the cells was statistically analyzed, showing a significant increase (e.g., ...). Figure 7 As shown in the figure, this indicates that QD-labeled integrins still have activation functions, and the labeled integrins can participate in the formation of adherents.

[0069] Comparative Example 1 Integrins were labeled using primary antibody and secondary antibody carrying fluorescent protein.

[0070] The experimental conditions were the same as in Example 2, except that after labeling the integrin with a primary antibody, excess antibody was washed away, and then the antibody was ligated with a secondary antibody containing green fluorescent protein Alex488 (GFP). Imaging and analysis were then performed using 488nm excitation light. The results are as follows: Figure 8As shown, the left image is the imaging result of the comparative example, and the right image is the imaging result of the quantum dot-labeled integrin of the present invention. It can be seen that the imaging image of the fluorescent protein-labeled integrin (left image) is unclear and cannot distinguish individual integrins, while the imaging image of the quantum dot-labeled integrin of the present invention (right image) is very clear and can clearly distinguish individual integrins, thereby enabling the analysis of the diffusion trajectory of individual integrins.

[0071] Comparative Example 2 Studying epithelial-mesenchymal transition using the cadherin assay (immunoglucoprotein staining)

[0072] Cells were fixed at room temperature for 1 hour (4% formaldehyde solution), washed with PBS, and then perforated for 10 minutes (0.2% Triton X-100). After washing with PBS, cells were incubated with 1% BSA for 1 hour, followed by overnight incubation with anti-E cadherin monoclonal antibody (12 hours, 4°C). The next day, cells were washed with PBS, incubated with Alex488-anti-mouse IgG (1 hour, room temperature), and then phalloidin-594 was added for actin staining. After washing with PBS, images were formed. The results are shown in the figure. Figure 9 The results showed that after EMT (right figure), the adhesion protein cadherin at the cell-cell boundary decreased. While this method can also distinguish between E-type and M-type cells, it requires cell fixation, cannot perform live-cell experiments, and is very time-consuming, requiring overnight incubation (more than 12 hours). The steps are cumbersome and unsuitable for rapid analysis of cell state. In contrast, the method of this invention is performed in live cells and typically takes less than one hour, greatly improving detection efficiency.

Claims

1. A method for detecting epithelial-mesenchymal transition of a cell, the method comprising the following steps: (1) incubating a single-biotin-labeled integrin antibody with a cell to be detected to specifically recognize and bind to integrins on the cell membrane surface of the cell, and removing unbound single-biotin-labeled integrin antibody after the incubation is completed; wherein the single-biotin-labeled integrin antibody is a single-biotin-labeled anti-β1 integrin antibody; (2) adding a streptavidin-labeled fluorescent probe to the product of step (1) and incubating, removing unreacted excess streptavidin-labeled fluorescent probe after the reaction, and obtaining fluorescent probe-labeled integrins; wherein the fluorescent probe is a quantum dot; (3) imaging the fluorescent probe of the fluorescent probe-labeled integrins obtained in step (2) to detect changes in the average diffusion speed of the fluorescent probe-labeled integrins relative to control cells, wherein an increase in the average diffusion speed of the fluorescent probe-labeled integrins relative to control cells indicates epithelial-mesenchymal transition of the cell, wherein the cell is selected from one or more of epithelial cells, tumor cells, and wherein the method is not used for diagnostic purposes. 2.The method of claim 1, wherein the average diffusion speed of the fluorescent probe-labeled integrins increases by 40%-172% relative to control cells. 3.The method of claim 1, wherein the average diffusion speed of the fluorescent probe-labeled integrins increases by 80%-172% relative to control cells. 4.The method of any one of claims 1-3, wherein the method further comprises a step of pretreating the cell before step (1). 5.The method of any one of claims 1-3, wherein the cell is an epithelial cell. 6.The method of any one of claims 1-3, wherein the cell is normal breast cell MCF10A or breast cell MCF10A after epithelial-mesenchymal transition. 7.The method of any one of claims 1-3, wherein the control cell is selected from a corresponding cell that has not undergone epithelial-mesenchymal transition. 8.The method of any one of claims 1-3, wherein the control cell is an epithelial cell. 9.The method of any one of claims 1-3, wherein the control cell is normal breast cell MCF10A. 10.The method of any one of claims 1-3, wherein in step (1), the incubation time is 10 to 18 minutes, and the incubation temperature is 0 to 4℃. 11.The method of any one of claims 1-3, wherein the incubation time is 15 minutes, and the incubation temperature is 0℃. 12.The method of any one of claims 1-3, wherein in step (2), the incubation time is 3 to 8 minutes, and the incubation temperature is 0 to 4℃. 13.The method of any one of claims 1-3, wherein in step (2), the incubation time is 5 minutes, and the incubation temperature is 0℃. ​ ​ ​ ​ ​ 14. The method of claim 12, wherein the quantum dots are selected from the group consisting of CdTe, CdSe, and CdSe / ZnS core-shell quantum dots.

15. The method of claim 12, wherein the quantum dots are CdSe / ZnS core-shell quantum dots.

16. The method of claim 12, wherein the quantum dots are selected from the group consisting of one or more quantum dots having a maximum emission wavelength of 525 nm to 800 nm.

17. The method of claim 12, wherein the quantum dots are quantum dots having a maximum emission wavelength of 600 nm to 800 nm.

18. The method of claim 12, wherein the quantum dots are quantum dots having a maximum emission wavelength of 655 nm.

19. The method of any one of claims 1-3, wherein in step (3), the imaging is at an emission wavelength of 525 nm to 800 nm.

20. The method of any one of claims 1-3, wherein in step (3), the imaging is at an emission wavelength of 600 nm to 800 nm.

21. The method of any one of claims 1-3, wherein in step (3), the imaging is at an emission wavelength of 655 nm.

22. The method of any one of claims 1-3, further comprising a step of centrifuging the fluorescent probe with streptavidin prior to step (2).

23. The method of claim 22, wherein the centrifuging is at 3000-7000 g for 2-5 minutes.

24. The method of claim 23, wherein the centrifuging is at 5000 g for 3-4 minutes.

25. Use of an integrin antibody with a single biotin and a fluorescent probe with streptavidin in the preparation of a kit for detecting epithelial-mesenchymal transition in a cell, wherein, the cell is selected from the group consisting of one or more of an epithelial cell, a tumor cell; the integrin antibody with a single biotin is an anti-βl integrin antibody with a single biotin; the fluorescent probe is a quantum dot.

26. A kit for detecting epithelial-to-mesenchymal transition of a cell, the kit comprising:

26. A kit comprising an integrin antibody with a single biotin and a fluorescent probe with streptavidin, wherein the cell is selected from the group consisting of one or more of an epithelial cell, a tumor cell; the integrin antibody with a single biotin is an anti-βl integrin antibody with a single biotin; the fluorescent probe is a quantum dot.

27. The kit of claim 26, wherein the quantum dots are selected from the group consisting of CdTe, CdSe, and CdSe / ZnS core-shell quantum dots.

28. The kit of claim 26, wherein the quantum dots are CdSe / ZnS core-shell quantum dots.

29. The kit of claim 26, wherein the quantum dots are selected from the group consisting of one or more quantum dots having a maximum emission wavelength of 525 nm to 800 nm.

30. The kit of claim 26, wherein the quantum dots are quantum dots having a maximum emission wavelength of 600 nm to 800 nm.

31. The kit of claim 26, wherein the quantum dots are quantum dots having a maximum emission wavelength of 655 nm.

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