A method for visualizing the recognition profile of rare single cells

Abstract

The present invention belongs to the field of liquid biopsy, and particularly relates to a method for visualizing the recognition profile of rare single cells, comprising the following steps: specifically staining the cell membranes of circulating tumor cells; adding a fluorescence enhancement reagent, which is an organic phase solution of a surfactant or an organic alcohol; performing microscopic imaging; converting the microscopic image into a digital photograph, and amplifying the digital signal in the digital photograph to obtain a visualized cell contour map. The present invention uses a surfactant or an organic alcohol to enhance the specific fluorescence on the cell surface, thereby improving the signal-to-noise ratio of the surface fluorescence signal; by using digital signal amplification technology, the visualization of the specific recognition profile of single cells is achieved, avoiding signal loss of the imaged cells; based on the fluorescence-visualized cell contour, the accuracy of capturing CTC counting and size estimation can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of liquid biopsy, and specifically relates to a method for visualizing the identification contour of a rare single cell, which is a method for visualizing the identification contour of a rare single cell based on combining fluorescence enhancement of cell membrane surface markers with digital signal amplification. Background Art

[0002] When the tissue cells of solid tumors undergo epithelial-mesenchymal transformation and fall off, the tumor cells will enter the venous blood in a single cell state and circulate in the body, namely circulating tumor cells (CTCs). They can attach to or be intercepted in remote organs, that is, tumor metastasis. Since CTCs accompany the occurrence and development of tumors and have a dose-response relationship with development, they are considered to be markers for early diagnosis and are comparable to the gold standard of clinical diagnosis, "tissue biopsy", and are also called liquid biopsy. Liquid biopsy usually has three basic steps: (1) obtaining CTCs from venous blood, including in vitro blood separation or capture of CTCs, and in vivo venous blood vessel capture of CTCs; (2) cell pretreatment before detection, such as cell fixation, blocking, membrane perforation, fluorescently labeled recognition ligands (such as antibodies, aptamers) to recognize cell membrane markers, nuclear staining, centrifugation, washing, etc. Among them, the recognition of cell membrane markers by fluorescently labeled recognition ligands achieves specific staining of the captured CTC membrane, namely "specific membrane staining", which is the key factor in determining whether the captured cells are CTCs; (3) microscopic imaging and microscopy. In order to ensure the accuracy of microscopic examination, when the pre-treated cell imaging, that is, the fluorescent dots in the photo, is judged as CTC, the laboratory and clinical practice usually require that three conditions be met at the same time, namely, the cell nucleus stained with DNA dye, the specific membrane staining can be observed outside the cell nucleus, and the cell size (usually 10-60μm) is significantly larger than the blood cell (usually 3-5μm). The latter two are dependent on the fluorescence intensity of the cell membrane-specific staining. If the fluorescence is weak, the specific membrane fluorescence of the imaged CTC cannot be effectively observed, and the CTC count based on the imaged cells will be low. At the same time, the cell scale cannot be estimated more accurately. The reason is that during microscopic examination, the cell image is often a fluorescent dot or circle. The size of the fluorescent dot is estimated based on the imaging scale to obtain the cell scale. This requires that the fluorescence intensity of the cell imaging dot must be sufficiently greater than the minimum detection limit of the eye. At the same time, a sufficient amount of markers distributed in large quantities on the entire cell membrane must be specifically identified to make the fluorescence of the imaging dot evenly distributed, avoiding incomplete fluorescent dots and irregular shapes, so as to ensure that the image contour is consistent with the cell contour of the CTC itself, so that the cell scale can be estimated more accurately based on the complete contour. Therefore, the accuracy of CTC counting and scale estimation during microscopic examination requires that the cell imaging needs to have a "complete cell contour that can be visualized". The root cause is that the fluorescence intensity of cell membrane-specific staining must be strong enough and significantly greater than the minimum detection limit of the eye.

[0003] The accuracy of CTC counting is the premise of CTC detection as a basis for early diagnosis of cancer. Since the number of CTCs is extremely rare and they are typical rare cells, the average value of detection in patients with advanced stage is usually less than 10, such as 5 for lung cancer (Emilsson, V., et al. Clin. Cancer Res., 2016, 22: 2197) and 5.5 for breast cancer (Nadia SZ, et al. Int. J. Oncol., 2012, 41: 1241), and the positive threshold is only 1 CTC. With such a low threshold and a very small number of cells, it is very easy to fail to detect CTCs under the microscope due to the low count of CTC microscopic imaging, making the diagnosis false negative. Therefore, enhancing the fluorescence intensity of cell membrane-specific staining to obtain a complete visual cell outline is the key to accurate CTC counting and accurate scale estimation.

[0004] Using a large number of specific expression proteins distributed on the surface of the CTC membrane as recognition receptors, with fluorescently labeled specific recognition ligands (such as antibodies and aptamers) acting on CTCs, the cell membrane can be specifically stained by forming a "receptor...ligand-fluorescent body" complex on the cell membrane surface. In theory, specifically stained fluorescent dots can be observed under a microscope, and the outline of the dots should be consistent with the actual outline of the CTC. Based on this, CTC counting can be performed based on specific fluorescent dots, and CTC scale estimation can be performed based on the complete outline of the dots. It is based on this that current laboratories and clinics have realized CTC counting and scale estimation under microscopy (Emilsson, V., et al. Clin. Cancer Res., 2016, 22: 2197; Li R, et al. ACS Applied Materials & Interfaces, 2018, 10: 16327).

[0005] However, the specific fluorescence of CTCs under the microscope is relatively weak, and the difference from the background fluorescence (including the fluorescence of the cell itself's substrate, miscellaneous spots, slides, etc.) is not large enough, that is, the signal-to-noise ratio is relatively low, and in some cases, fluorescence cannot even be observed; the fluorescent dots presented by many cells are incomplete and irregular in shape. There are two reasons for this. First, there are a large number of protein molecules on the cell membrane surface, and they have obvious matrix fluorescence, which must be deducted during microscopy, resulting in a significant reduction in the intensity of specifically recognized fluorescence. Moreover, the cell fluid is a heterogeneous suspension, and the intensity of specifically recognized fluorescence on the membrane surface is also uneven. Strong fluorescence can be effectively observed, while weak fluorescence cannot be effectively observed, which leads to the incompleteness of the fluorescent dots in cell imaging. Second, the fluorescence excitation light source of the inverted fluorescence microscope widely used in clinics and laboratories is usually a high-pressure mercury lamp with significantly lower energy than a laser. This can effectively reduce fluorescence quenching but also weakens the intensity of the excited fluorescence. This necessarily results in a significant undercount of CTCs based on the fluorescent dots of imaging cells and a significant deviation in cell size estimation. To solve the above problems, it is necessary to enhance the fluorescence intensity of the specific staining of the CTC cell membrane to increase the difference between specific fluorescence and background fluorescence, that is, to increase the signal-to-noise ratio, so that the difference between the fluorescence brightness of cell imaging and the background is obvious. After deducting the background (that is, there is no background fluorescence in the photo, only the specific fluorescence of the cells) during imaging observation, the remaining specific fluorescence of the cells has sufficient intensity, which is a prerequisite for visualizing the complete cell contour. When the remaining intensity is significantly greater than the minimum detection limit of the eye (that is, the lowest intensity that can be observed), visualization is achieved, and direct microscopy observation can be carried out; otherwise, visualization cannot be achieved, and microscopy is still inaccurate. In fact, more CTCs belong to the latter case. Although the fluorescence intensity at this time does not reach the minimum detection limit of the eye, it is significantly higher than the background fluorescence, that is, the signal-to-noise ratio is significantly increased. To sum up, to fully visualize the extremely small number of captured CTCs, two problems need to be solved. First, enhance the specifically recognized fluorescence of the cell membrane and improve the signal-to-noise ratio of specific fluorescence; second, how to make the intensity of the high-signal-to-noise ratio signal significantly higher than the minimum detection limit of the eye, so as to present the complete cell contour and achieve cell visualization. There are many methods to enhance fluorescence, and micelle-enhanced fluorescence is a common strategy. It involves encapsulating hydrophobic fluorophores in the inner layer of micelles to form a hydrophobic spherical core, creating a hydrophobic microenvironment for the fluorophores to enhance fluorescence, while the outer layer of the micelles forms a hydrophilic spherical shell. There are mainly two types of methods. First, amphiphilic block copolymers have both non-fluorescent hydrophilic ends and fluorescent lipophilic ends. Using hydrophobic interactions, hydrophilic spherical micelle nanoparticles with the hydrophilic ends facing outward and the fluorescent lipophilic ends facing inward are formed in an aqueous solvent. Second, non-fluorescent surfactants also have hydrophilic and lipophilic ends and can also form hydrophilic spherical micelles in an aqueous solvent. During the formation of micelles, hydrophobic fluorescent molecules are fixed into the hydrophobic spherical core of the micelles by encapsulation. They have three common characteristics.First, enhance the fluorescence of free fluorescent molecules in the solution, including encapsulated free fluorescent small molecules and free fluorescent polymer molecules aggregated based on hydrophobic interactions; second, the phosphor is fixed in the hydrophobic spherical core, and the entire micelle is a hydrophilic spherical nanoparticle; third, a large number of fluorophores or fluorescent molecules are aggregated in the core of the spherical micelle. The prepared fluorescent-enhanced micelle nanoparticles are used as fluorescent signal bodies to label specific recognition ligands (such as antibodies, aptamers), and then used to recognize the surface markers of CTC membranes to achieve enhanced fluorescence for CTC membrane recognition. This strategy of "first preparing fluorescent-enhanced micelle nanoparticles, then labeling recognition ligands, and finally recognizing receptors on the CTC membrane" to achieve enhanced fluorescence for CTC specific recognition. The hydrophobic microenvironment required to enhance the fluorescence of the phosphor is provided by the micelle nanoparticles formed by the aggregation of the hydrophobic ends of surfactants, and there will inevitably be four aspects of defects caused by the micelle nanoparticles themselves. (1) After the recognition ligand is labeled with fluorescent micelle nanoparticles, since the recognition ligand is a molecule and its volume is much smaller than the micelle nanoparticles connected to it, when the recognition ligand recognizes the receptor on the cell membrane surface, the micelles connected to it will generate a large steric hindrance, and the recognition ability will be significantly weaker than that of the recognition ligand labeled with small-volume fluorescent molecules, and the specific fluorescence of recognition will be weakened, which partially offsets the fluorescence of the cell membrane enhanced by the micelle nanoparticles. (2) The process of preparing fluorescent-enhanced nanoparticles is relatively complex, including connecting fluorescent small molecules with long-chain macromolecules to prepare fluorescent amphiphilic long-chain macromolecules, connecting reactive groups to the fluorescent amphiphilic long-chain macromolecules to achieve the labeling of recognition ligands, stabilizing the structure of micelle nanoparticles, and dialysis separation of excess fluorescent small molecules through membranes, etc.; (3) The encapsulation rate of fluorescent small molecules in micelle nanoparticles is uneven, and even some nanoparticles do not encapsulate fluorescent small molecules, resulting in uneven fluorescence intensity of the labeled recognition ligands or even no fluorescence; (4) The batch-to-batch difference in the fluorescence intensity of micelle nanoparticles is extremely large, resulting in poor reproducibility of the recognition fluorescence of cells. It is precisely for the above reasons that although there are a large number of literature reports on fluorescent-enhanced nanoparticles, it is difficult to find their practical applications in clinical detection.

[0006] From the perspective of application, since the enhancement of fluorescence by micelle nanoparticles is to change the microenvironment of fluorescent signal molecules, thereby achieving physical enhancement of the signal, the ability of this type of signal enhancement (i.e., the enhancement factor) will be restricted by many factors. For example, the larger the nanoparticle, the more fluorescent molecules it encapsulates, and the greater the increase in fluorescence. However, when the recognition ligand labeled with fluorescent particles recognizes the receptor on the cell membrane, the large-volume fluorescent particles will generate a large steric hindrance, and the recognition ability will decrease, and the fluorescence intensity on the cell membrane will instead decrease, forming a mutually restrictive relationship. Therefore, usually the specific recognition fluorescence of the cell membrane is still weak. After subtracting the fluorescence of the cell matrix, the brightness of the imaged cells is low, and the complete contours of all cells cannot be clearly observed, and even some cells cannot be observed with fluorescence. Summary of the Invention

[0007] The present invention aims to solve the above problems and provides a method for visualizing the recognition profile of rare single cells. Based on the combination of fluorescence enhancement of cell membrane surface marker recognition and digital signal amplification, and based on the fluorescence-visualized cell profile, the accuracy of capturing CTC count and scale estimation can be achieved.

[0008] According to the technical solution of the present invention, the method for visualizing the recognition profile of rare single cells includes the following steps:

[0009] S1: Perform cell membrane fluorescence staining for specific recognition of cell membrane markers of rare single cells to obtain cell solution I with water or aqueous solution as the solvent;

[0010] S2: Add a fluorescence enhancement reagent to the cell solution I to obtain cell solution II. The fluorescence enhancement reagent is an organic phase solution of a surfactant or a liquid organic alcohol;

[0011] S3: Perform microscopic imaging on the cell solution II;

[0012] S4: Convert the microscopic image photo into a digital photo, and amplify the digital signal in the digital photo to obtain a visualized cell contour map.

[0013] Before the present invention completes the conventional cell pretreatment before microscopy, including cell fixation, blocking, membrane perforation, specific membrane staining, nuclear staining, centrifugation, washing, etc., adding a simple operation can achieve the enhancement of specific recognition fluorescence, that is, directly drop an organic phase solution of a surfactant, such as an N,N-dimethylformamide (DMF) solution of surfactant Tween-80 (an aqueous solution of Tween-80 cannot be used, such as a PBS solution of Tween-80, a distilled aqueous solution of Tween-80) into the pretreated cell solution, and then perform microscopy imaging according to the conventional method, including adding the cell solution to the glass slide, standing, and microscopy; or directly drop a liquid organic alcohol with excellent water solubility, such as ethanol, onto the cell solution on the glass slide, and directly perform microscopy after standing. Compared with the current conventional method, only one step of "dropping an organic phase solution of a surfactant or a liquid organic alcohol with excellent water solubility" is added to the cell solution after specific recognition, and other complex operations such as centrifugation and dialysis do not need to be added, and microscopy can be performed according to the current method. Therefore, the method is simple and consistent with the current microscopy method, and is easy to be popularized and applied in laboratories and clinics. Secondly, there is no need to prepare fluorescent micelle nanoparticles with complex processes. Of course, there are no structural defects of fluorescent micelle nanoparticles, such as uneven encapsulation rate and poor process reproducibility.

[0014] The present invention constructs a hydrophobic microenvironment that is different in principle from micelle-enhanced fluorescence. After cells are specifically stained with membrane fluorescence, a "receptor... ligand-fluorophore" complex is formed on the cell membrane surface. The fluorophore extends outward away from the cell membrane, with the fluorophore group located at the distal end, like an "incompletely nailed nail on the cell membrane", and the fluorescent molecule is like the head of the nail. At this time, the fluorophore is in a constrained state, unlike the fluorescent molecules in general micelle-enhanced fluorescence, which are in a free state. For the recognized cells, whether an organic phase solution of a surfactant is dropped onto the cell surface or a liquid organic alcohol with excellent water solubility is added, it also constructs a hydrophobic microenvironment for the fluorophore on the membrane.

[0015] When an organic phase solution of a surfactant is dropped, the surfactant is uniformly distributed in the solution in molecular form in the "organic phase" solvent. When dropped into the suspension of recognized cells or the cell droplet on a glass slide, since the solvent of the cells is an "aqueous solvent", such as distilled water, PBS, or DPBS, providing a hydrophilic large environment, the strong hydrophobicity of the "fluorophore group of the fluorescent molecule like the nail head" will induce the hydrophobic ends of the surfactants free in water to accumulate towards it and the hydrophilic ends to extend outward, forming an aggregate like an "umbrella-shaped hat" that buckles on the fluorophore outside the cell membrane. We call this aggregate the surface umbrella-shaped micelle. At this time, the fluorescent molecule is surrounded by the umbrella-shaped micelle and the cell membrane surface protein under the micelle, forming a closed space, which also constitutes a hydrophobic microenvironment for the fluorescent molecule. The reason why a hydrophobic microenvironment of a normal spherical micelle cannot be formed is that the "receptor... ligand" in the "receptor... ligand-fluorophore" complex on the cell membrane and the "cell membrane" impose spatial restrictions on it, causing the umbrella-shaped micelle to "open a mouth", and this "opening" is exactly blocked by the cell membrane surface protein, thus forming a hydrophobic microenvironment for the fluorophore group in the core.

[0016] When a liquid organic alcohol with excellent water solubility is added to the droplet of recognized cells, due to the good water solubility of the liquid organic alcohol, it is very easy to mix with a large amount of water in the cell membrane. The organic alcohol with "more alcohol and less water" is very volatile, resulting in dehydration of the cell membrane, the membrane producing wrinkles, making the membrane protein molecules approach each other and fully expose. On the one hand, the dehydration of the membrane increases the membrane hydrophobicity, providing a hydrophobic microenvironment for the fluorescent molecules on the surface; on the other hand, the fluorophores specifically recognized and located on the membrane proteins approach each other due to the membrane wrinkles, and the hydrophobic fluorophore groups of the fluorophores also provide a hydrophobic microenvironment for each other due to aggregation.

[0017] For the cell imaging photos obtained by microscopy, there are still problems such as "the brightness of the specific fluorescence on the imaged cell membrane is still low, and the non-uniformity of the brightness, making it impossible to clearly observe the complete cell contour, and even some cells cannot observe fluorescence". For general cell imaging, this is already sufficient to achieve relatively accurate cell imaging detection. The reason is that conventional cell imaging microscopy generally needs to check two parameters, whether the cell is a target cell and the number of target cells. Since there are a large number of conventional cells, dozens, even hundreds or thousands in one field of view, and the total number of cells that can be microscopically examined is large, a small number of cells with weak fluorescence not being counted will not significantly affect the accuracy of the results, nor will it lead to false negatives. In the cells after specific recognition and those with increased fluorescence, as long as specific fluorescence can be observed, it can be determined as a target cell. Since there is no need to estimate the cell scale, there is no need to "present a complete cell contour". For rare cells CTC, when microscopically examining the imaged cells, it is necessary to require all cells to be completely counted and the cell contour to be complete and clearly visible in order to achieve a relatively accurate estimate of the cell scale. Therefore, through the above-mentioned signal of enhanced specific fluorescence, the requirements for CTC counting and scale estimation still cannot be met. By dropping an organic phase solution or a liquid organic alcohol of a surfactant, the absolute value of the specific recognition fluorescence intensity of the cell membrane is increased, and at the same time, the relative value, that is, the signal-to-noise ratio, is also increased. After digitizing the photo, the digital signal is amplified by a certain multiple. Since the signal-to-noise ratio is increased, the intensity difference between the specific fluorescence and the non-specific matrix fluorescence is increased. Although the amplification multiple determined by the criterion that the matrix fluorescence cannot be observed remains unchanged, when amplified by the same multiple, since the absolute value of the specific fluorescence intensity is increased, the amplified absolute value is significantly increased, making its intensity far greater than the lowest detection limit of the eye, and thus the complete contour of all cells can be visualized. Specifically, the rare single cell can be a circulating tumor cell (CTC) or a fetal-derived cell in maternal blood.

[0018] In one embodiment, in the step S1, the rare single cell is CTC, the specific membrane protein EpCAM of CTC is used as the recognition receptor, and the EpCAM nucleic acid aptamer (Apt-FAM) labeled with carboxyfluorescein (FAM) is used as the recognition ligand. Apt-FAM specifically recognizes the cell membrane protein EpCAM, localizes Apt-FAM on the cell membrane, and the labeled fluorescence FAM therein is far from the cell surface and is distributed in large amounts on the surface.

[0019] Furthermore, the solvent of the cell liquid I is distilled water or an aqueous solution such as PBS or DPBS.

[0020] Further, before the step S1, an operation of fixing the rare single cells is also included; through fixation, the cell membrane markers (proteins) of the rare single cells are fully exposed, facilitating the recognition by ligands (such as antibodies, aptamers), thereby improving the cell membrane specific fluorescence staining effect.

[0021] Specifically, the rare single cells are fixed with a fixing solution, and the fixing solution includes acetone and paraformaldehyde solution.

[0022] Further, before the step S1, an operation of blocking the non-specific sites on the cell membrane of the circulating tumor cells is also included.

[0023] Specifically, the non-specific sites on the cell membrane of the circulating tumor cells are blocked by adding a random base library.

[0024] Further, in order to localize the stained cell membrane to determine whether it is outside the cell nucleus, an operation of staining the cell nucleus of the circulating tumor cells is also included in the step S1. Specifically, the cell nucleus can be stained with Hoechst 33342.

[0025] Further, in the step S2, the fluorescence enhancing reagent is added in a manner of directly adding to cell solution I (that is, first adding the fluorescence enhancing reagent to cell solution I, and then transferring it to the carrier for microscopic imaging), or dropping it onto cell solution I on the carrier (that is, first transferring cell solution I to the carrier, and then adding the fluorescence enhancing reagent for microscopic imaging), and the carrier includes a glass slide, a culture dish, or various well plates.

[0026] Specifically, when the fluorescence enhancing reagent is an organic phase solution of a surfactant, both addition methods are applicable. When the fluorescence enhancing reagent is a liquid organic alcohol, to improve the fluorescence enhancing effect, the preferred method is to drop it onto cell solution I on the carrier. When the fluorescence enhancing reagent is an organic phase solution of a surfactant and the addition method is to directly add it to cell solution I, it is necessary to ensure that the solvent of cell solution I is water or an aqueous solution (such as distilled water, PBS, or DPBS, etc. are all acceptable).

[0027] Further, the surfactant is selected from cationic surfactants, anionic surfactants, or non-ionic surfactants. Preferably, it is a Tween series non-ionic surfactant, such as Tween-20, Tween-60, or Tween-80, and more preferably Tween-80.

[0028] Further, the concentration of the surfactant in cell solution II is 1 - 16 mmol / L, preferably 7 - 14 mmol / L.

[0029] Specifically, the concentration of the surfactant in the organic phase solution of the surfactant is 3-100 mmol / L, and the volume ratio of the organic phase solution of the surfactant to the cell liquid I is 1:3-6.

[0030] Further, the liquid organic alcohol is selected from methanol, ethanol, propanol or isopropanol.

[0031] Further, in the step S3, the specific operation of microscopic imaging can be as follows: taking a bright-field photo; taking fluorescence photos of nuclear staining and membrane staining respectively under the dark fields of ultraviolet and blue light; overlapping the bright-field photo and the fluorescence photos. Among them, during actual detection, only the bright-field photo and the membrane staining fluorescence photo under the blue light dark field need to be taken and overlapped to judge whether there is no leakage in membrane staining and whether the simultaneously presented membrane contours are of the same size.

[0032] Further, in the step S4, the photos of microscopic imaging are converted into digital photos by using a compiled program, and then the digital photos are subjected to specific digital signal amplification or non-specific digital signal amplification to obtain a visualized cell contour map.

[0033] Further, the non-specific digital signal amplification is to amplify all the digital signals in the photo, and the amplification multiple is based on the standard that the matrix fluorescence cannot be observed, and at the same time, the cell contour is clearly visible.

[0034] Further, the specific digital signal amplification is to amplify the signals with a signal-to-noise ratio greater than 3, and the amplification multiple is based on the standard that the cell contour is clearly visible.

[0035] The technical solution of the present invention has the following advantages compared with the prior art:

[0036] The present invention uses a surfactant or a liquid organic alcohol to enhance the specific fluorescence on the cell surface, thereby improving the signal-to-noise ratio of the surface specific fluorescence signal; by using the digital signal amplification technology, the membrane fluorescence signal of the imaged cell is amplified, and the contour of the imaged cell is presented completely and clearly, realizing the visualization of the specific recognition contour of a single cell and avoiding the signal loss of the imaged cell; based on the fluorescence-visualized cell contour, the accuracy of capturing CTC counting and scale estimation can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic diagram of the principle (the first row) before (left column) and after (right column) the formation of single-cell surface umbrella-shaped micelles induced by a fluorophore in Example 2, and SEM (the second and third rows) imaging.

[0038] Figure 2 It is the microscopic imaging of a single cell with specific recognition (left) and its digital signal amplified cell photo (right) in Example 2 (scale bar: 10 μm).

[0039] Figure 3 Single-cell microscopic imaging of specific recognition without fluorescence enhancement (upper) and with fluorescence enhancement in Example 2 (lower) (left), and cell photos with digital signal amplification (right) (scale bar: 10 μm).

[0040] Figure 4 Single-cell microscopic imaging of fluorescence enhancement of Tween-20 (left), Tween-60 (middle), and Example 2 (right) (upper), and cell photos with digital signal amplification (lower) (scale bar: 10 μm).

[0041] Figure 5 Single-cell microscopic imaging of fluorescence enhancement of 7.5 mmol / L (upper) and 13 mmol / L (lower) Tween-80 (left), and cell photos with digital signal amplification (right) (scale bar: 10 μm).

[0042] Figure 6 Single-cell microscopic imaging of specific recognition without fluorescence enhancement (upper) and with fluorescence enhancement in Example 5 (lower) (left), and cell photos with digital signal amplification (right) (scale bar: 10 μm). Detailed implementation manners

[0043] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0044] The reagents and instruments in the following embodiments are as follows:

[0045] EpCAM nucleic acid aptamer (Apt-FAM, sequence: 5′FAM-T TTT TTT TTT CAC TACAGA GGT TGCGTC TGT CCC ACG TTG TCATGG GGG GTT GGC CTG-3′, Sangon Biotech); 48-base random library (Sangon Biotech); trypsin cell digestive solution, penicillin-streptomycin double antibody, medium RPMI-1640, fetal bovine serum (Beyotime Biotechnology); MCF-7 human breast cancer cells (School of Pharmacy, Soochow University), and other reagents are all of analytical purity. Inverted fluorescence microscope (WMF-3650, Shanghai Wumo Optical Instrument Co., Ltd.).

[0046] Example 1: Cultivation and digestion of cells

[0047] For the convenience of example description, very low-density single cells obtained by culturing, digesting, and resuspending cells are used to simulate a very small amount of CTCs captured.

[0048] The breast cancer cell line MCF-7 was placed in complete RPMI-1640 medium and incubated at 37°C with 5% CO2. When it reached the logarithmic growth phase, the culture flask was taken out, the culture medium was discarded, and 3 mL of DPBS solution was added for washing, repeating twice. Then, 1 mL of trypsin was added and digested at 37°C for 3 minutes. The digestion solution was discarded, 3 mL of complete medium was added, and the adherent cells were pipetted and blown. The cell suspension was aspirated and added to a 15 mL centrifuge tube, centrifuged at 1500 rpm / min for 3 minutes, the supernatant was discarded, and then it was resuspended with 3 mL of DPBS (isotonic phosphate buffer solution) for standby.

[0049] Example 2: Visualization of the recognition profile of rare single cells

[0050] 1. Cell pretreatment

[0051] Take 1 mL of the suspension (obtained in Example 1) and place it in a brown centrifuge tube, centrifuge at 3000 rpm / min for 3 minutes, discard the supernatant, resuspend with 1 mL of DPBS, centrifuge again and discard the supernatant, add 300 μL of acetone to resuspend the cells, after 10 minutes centrifuge at 3000 rpm / min for 3 minutes, discard the supernatant, and 3 tubes of fixed cells can be obtained. Two of them are stored at 4°C for standby. To another tube of fixed cell pellet, add 1 mL of triple-distilled water to resuspend, centrifuge and discard the supernatant to wash away the residual acetone. Resuspend with 1 mL of 1% Triton X-100 (polyethylene glycol octyl phenyl ether) solution, after 10 minutes centrifuge and discard the supernatant, then add 1 mL of triple-distilled water to resuspend, centrifuge and discard the supernatant to wash away the residual Triton X-100, achieving cell membrane permeabilization. Take 20 μL of triple-distilled water to resuspend the cells, add 10 μL of 10 μM / L random base library, after 30 minutes add 500 μL of triple-distilled water, centrifuge and discard the supernatant, achieving the blocking of non-specific sites on the cell membrane, and the cells are ready for use.

[0052] 2. Specific cell membrane staining

[0053] Take 20 μL of triple-distilled water to resuspend the cells, add 10 μL of Apt-FAM aptamer solution (10 μM / L) under light protection conditions, pipette and mix evenly, stain for 30 minutes, and pipette and mix once every 5 minutes during this period. Then, add 500 μL of triple-distilled water, pipette and mix evenly, centrifuge, discard the supernatant, resuspend the cells with 1 mL of triple-distilled water to obtain cells with membrane-specific staining. Add 8 μL of DMSO (dimethyl sulfoxide) solution of Hoechst 33342 (2 mg / mL), pipette and mix well, rotate and mix for 15 minutes. Centrifuge, discard the supernatant, resuspend with 1 mL of triple-distilled water, and repeat the operation 3 times to wash away the excess Hoechst 33342 fluorescent dye to avoid significant background fluorescence interference, obtaining a cell suspension with membrane staining and nuclear staining.

[0054] 3. Fluorescence Enhancement of Specific Staining (Surfactant) and Microscopic Imaging (Method of Adding to Cell Suspension)

[0055] Centrifuge the cell suspensions stained for the membrane and nucleus, and discard the supernatant. Quickly add 500 μL of triple-distilled water, pipette to resuspend the cells, and then quickly add 100 μL of a DMF solution of Tween-80 (80 mmol / L) using a pipette. Then, pipette at the bottom of the test tube to mix it quickly and evenly, and let it stand for 20 min to obtain cells with enhanced membrane fluorescence. Take 100 μL of the cell suspension on a glass slide and let it stand for about 5 min to ensure that the cells settle on the glass slide. Observe whether the cell morphology is intact under bright field and take bright field photos. Then, take fluorescence photos of the nucleus and membrane staining under dark field with violet light (exposure time 800 ms) and blue light (exposure time 2000 ms) respectively. Use ImageView software to overlap the above three types of photos to obtain an overlapping photo of the cells to be tested.

[0056] 4. Fluorescence Signal Amplification of Specific Recognition of Cell Membrane (Non-Specific Signal Amplification)

[0057] Use a program self-written with Matlab software to obtain digital photos from the fluorescence photos taken under blue light dark field (fluorescence photos of membrane-specific staining). Amplify all digital signals in the digital photos by a certain multiple, with the standard that the matrix fluorescence cannot be observed, and at the same time, taking into account that the cell outline is clearly visible, thus obtaining a visualized cell outline diagram.

[0058] Example 3: Visualization of the Recognition Outline of Rare Single Cells

[0059] On the basis of Example 2, replace step 3 with:

[0060] 3'. Fluorescence Enhancement of Specific Staining (Absolute Ethanol) and Microscopic Imaging (Method of Adding to Cell Suspension)

[0061] Centrifuge the cell suspensions stained for the membrane and nucleus, and discard the supernatant. Quickly add 500 μL of absolute ethanol. Then, pipette at the bottom of the test tube to mix it quickly and evenly, and let it stand for 20 min to obtain cells with enhanced membrane fluorescence. Take 100 μL of the cell suspension on a glass slide and let it stand for about 5 min to ensure that the cells settle on the glass slide. Observe whether the cell morphology is intact under bright field and take bright field photos. Then, take fluorescence photos of the nucleus and membrane staining under dark field with violet light (exposure time 800 ms) and blue light (exposure time 2000 ms) respectively. Use ImageView software to overlap the above three types of photos to obtain an overlapping photo of the cells to be tested.

[0062] Example 4: Visualization of the Recognition Outline of Rare Single Cells

[0063] On the basis of Example 2, replace step 3 with:[[]]

[0064] 3'. Fluorescence Enhancement of Specific Staining (Surfactant) and Microscopic Imaging (Method of Adding to Cell Droplets on Slide)

[0065] Take 100 μL of the cell suspension stained with membrane and nucleus and place it on a slide. Let it stand for about 5 min to ensure that the cells settle on the slide. Add a DMF solution of Tween-80 (80 mmol / L) dropwise to the cell droplets on the slide. After standing for 5 - 8 min, observe whether the cell morphology is intact under bright field and take bright field photos. Then, take fluorescence photos of nucleus staining and membrane staining under dark field of violet light (exposure time 800 ms) and blue light (exposure time 2000 ms) respectively. Overlap the above three types of photos with ImageView software to obtain the overlapped photos of the cells to be detected.

[0066] Example 5: Visualization of the Recognition Profile of Rare Single Cells

[0067] On the basis of Example 2, replace step 3 with:[[]]

[0068] 3'. Fluorescence Enhancement of Specific Staining (Absolute Ethanol) and Microscopic Imaging (Method of Adding to Cell Droplets on Slide)

[0069] Take 100 μL of the cell suspension stained with membrane and nucleus and place it on a slide. Let it stand for about 5 min to ensure that the cells settle on the slide. Add absolute ethanol dropwise to the cell droplets on the slide. After standing for 5 - 8 min, observe whether the cell morphology is intact under bright field and take bright field photos. Then, take fluorescence photos of nucleus staining and membrane staining under dark field of violet light (exposure time 800 ms) and blue light (exposure time 2000 ms) respectively. Overlap the above three types of photos with ImageView software to obtain the overlapped photos of the cells to be detected.

[0070] Example 6: Visualization of the Recognition Profile of Rare Single Cells

[0071] On the basis of Example 2, replace step 4 with

[0072] 4. Fluorescence Signal Amplification of Specific Recognition of Cell Membrane (Specific Signal Amplification)

[0073] Use the program self-compiled by Matlab software to obtain digital photos from the fluorescence photos taken under blue light dark field (fluorescence photos of membrane-specific staining). Amplify the signals with a signal-to-noise ratio greater than 3 in the digital photos, and the amplification factor is based on the standard that the cell contour is clearly visible, that is, the visualized cell contour map is obtained.

[0074] Result Analysis

[0075] 1. As Figure 1As shown in the figure, by analyzing the principle of fluorescence enhancement of single-cell surface recognition in Example 2, it can be seen that:

[0076] Since specific protein EpCAM is overexpressed on the surface of tumor cell membranes, such as human breast cancer cell MCF-7, when a fluorescently labeled EpCAM ligand, such as a nucleic acid aptamer FAM-Apt labeled with FAM, contacts the tumor cells, the EpCAM ligand will specifically recognize the EpCAM receptors abundantly present on the cell membrane, forming a large number of distributed receptor-ligand complexes EpCAM···Apt-FAM on the cell membrane, causing a large amount of labeled fluorescence FAM to be distributed on the cell membrane surface. Under the fluorescence field, the membrane fluorescence contour displayed is the contour of the tumor cells, as shown in the left figure of the first row in Figure 1 As shown. Since Tween is a non-ionic surfactant with non-ionized hydrophilic and hydrophobic ends, when it contacts the receptor-ligand complex EpCAM···Apt-FAM on the cell membrane, it will not accumulate on the cell membrane due to charge interactions. The hydrophobic end of Tween will accumulate on the cell in two ways due to hydrophobic interactions. First, due to the hydrophobicity of the cell membrane, the hydrophobic chain of Tween lies flat on the membrane, which does not interact with the fluorophore FAM. Second, when Tween contacts the fluorophore of the FAM molecule in the receptor-ligand complex EpCAM···Apt-FAM, the hydrophobicity of the fluorophore will induce the hydrophobic end of Tween to accumulate around FAM due to hydrophobic interactions. At the same time, FAM is labeled with a nucleic acid aptamer of dozens of bases (such as 58 bases in this application), which will move FAM away from the cell membrane. If the labeled recognition ligand is an antibody, it will also move FAM away from the cell membrane due to the spacer arm effect of the antibody macromolecule. This provides enough space for the accumulation of Tween, thus forming an umbrella-shaped micelle around FAM, as shown in Figure 1 the right figure of the first row in. Since the umbrella-shaped micelle is buckled on the cell membrane, the membrane protein below just seals the opening of the umbrella-shaped micelle, constructing a hydrophobic microenvironment for the fluorophore FAM therein. It also exhibits the property of enhancing fluorescence of a spherical micelle usually composed entirely of surfactants, thus realizing the enhancement of specific recognition fluorescence of the cell membrane. From the SEM characterization, umbrella-shaped micelle nanoparticles can be observed on the surface of MCF-7 cells with enhanced specific fluorescence by Tween ( Figure 1 right, 3rd row), and there are a large number of surface nanoparticles ( Figure 1 right, 2nd row); for cells without enhanced specific fluorescence by Tween, the surface contour is clear and no umbrella-shaped micelle nanoparticles are found ( Figure 1 left, 2nd and 3rd rows), indicating that the umbrella-shaped micelle nanoparticles are formed by Tween.

[0077] 2. As shown in Figure 2As shown in the cell photos of single-cell microscopic imaging (fluorescence) with specific recognition and its digital signal amplification in Comparative Example 2, it can be seen that:

[0078] Since each specific recognition site on the surface of CTCs can only form a surface ligand-receptor fluorescent complex EpCAM···Apt-FAM with one Apt-FAM, even after the fluorescence is enhanced by the umbrella-shaped micelles formed by Tween-80, it often shows weak fluorescence under a fluorescence microscope. Coupled with the non-uniformity of the cell suspension, the cell contours that are usually weakly fluorescent and have incomplete contours ( Figure 2 left). This will inevitably lead to fewer CTCs detected by microscopy, even significantly fewer than the number of captured CTCs, which may be an important factor leading to false negatives. For single-cell microscopic imaging with specific recognition ( Figure 2 left), it can be found that the FAM fluorescence of the cells (actually green) is weak. A small number of cells can observe a complete and brighter green circle with a scale of about 10 μm, which is the visible complete cell contour. More are circles with weaker brightness, even incomplete cell contours with weaker brightness (cell imaging within the rectangular frame in the figure), and cells that cannot be effectively observed (cell imaging within the circle in the figure). When the digital signal is amplified ( Figure 2 right), the brightness of all cells is significantly increased, and the cell contours with a size of about 10 μm are completely visualized. In particular, the three cells that could not be effectively observed ( Figure 2 the cells within the circle in the left figure) also achieve the visualization of the complete contour, which provides ideal cell photos for accurate counting of the imaged cells and cell size estimation based on the complete contour. Of course, in the photo with digital signal amplification, a large number of high-brightness noise points appear. Because the digital signal amplification adopted amplifies all signals of the microscopic imaging, including the fluorescence signal, background signal, and noise signal of the cells, by the same multiple, that is, non-specific signal amplification, resulting in a significant increase in the brightness of the background signal and noise signal. However, this does not affect the counting and size estimation of the imaged cells. Because the size of the green circles of the cells is relatively consistent and much larger than the high-brightness noise points, and the background signal is usually extremely weak, it can be made weaker than the detection limit by selecting the signal amplification multiple and thus cannot be effectively observed.

[0079] 3. As Figure 3 shown, by comparing the cell photos of single-cell microscopic imaging (fluorescence) with specific recognition in Example 2 and that without fluorescence enhancement and its digital signal amplification, it can be seen that:

[0080] The surfactant Tween enhances the fluorescence of the labeled fluorescent molecule FAM on the ligand-receptor complex EpCAM···Apt-FAM on the cell surface. This is specific fluorescence enhancement, which will increase the difference between cell fluorescence and background fluorescence, thereby increasing the signal-to-noise ratio of imaging detection. This is a prerequisite for whether digital signal amplification can visualize the complete contour. See Figure 3 . It can be found that when Tween-enhanced specific recognition fluorescence is not implemented ( Figure 3 upper left), only 1 cell with extremely weak fluorescence and an incomplete contour can be seen. After signal amplification ( Figure 3 upper right), the complete contour of this cell can be visualized, while the fluorescence contours of other cells cannot be visualized. See the white circle with a scale of about 10 μm in the square in the figure (the green fluorescence of FAM not observed). After implementing Tween-enhanced specific recognition fluorescence ( Figure 3 lower left), the number of fluorescent cells with complete contours that can be visualized increases significantly, but the fluorescence signal is still weak and the brightness of the cell contours is still low; at the same time, there are still cells with extremely weak fluorescence and incomplete contours (2 cells within the circle). When the signal is amplified, the fluorescence brightness of the cells increases significantly, and all cells show high-brightness, visualizable, complete cell contours, and there are no longer cells that cannot be visualized by fluorescence ( Figure 3 lower right).

[0081] 4. As Figure 4 shown, when Tween-80 in Example 2 is replaced with Tween-20 and Tween-60 respectively, it can be known that the fluorescence of the latter two is extremely weak, and after signal amplification, the fluorescence of some cells still cannot be observed ( Figure 4 the white circles in the lower left and middle figures, the green fluorescence of FAM not observed), while after signal amplification of the cell fluorescence enhanced by Tween-80, all fluorescent cells can be "clearly" observed ( Figure 4 lower right). Therefore, Tween-80 is preferably used.

[0082] 5. As Figure 5 shown, by adjusting the concentration of Tween-80 in Example 2, it can be found that as the concentration of Tween-80 in the cell solution increases, the fluorescence signal becomes stronger; in the imaging with 7.5 mmol / L (the concentration of Tween-80 in the cell solution), the cell fluorescence is extremely weak ( Figure 5 upper left), while at 13 mmol / L, the cell fluorescence is significantly stronger than the former ( Figure 5 lower left). When the concentration continues to increase, turbidity will appear in the cell suspension, which is caused by the poor solubility of Tween-80 in aqueous solution. In the imaging with fluorescence enhancement at 13 mmol / L, all cells can be "clearly" observedFigure 5 lower right), while in the imaging with enhanced fluorescence at 7.5 mmol / L, there are still some cell fluorescences that cannot be observed ( Figure 5 upper right)

[0083] 6. As Figure 6 shown, the cell membrane fluorescence specifically recognized is weak when ethanol anhydrous enhancement is not implemented (upper left). There are also extremely weak cell fluorescences that are difficult to observe (as shown by the boxes in the figure), and even fluorescences that are basically unobservable (as shown by the circles in the figure). After digital amplification of the signal (upper right), most cell outlines can be clearly and completely observed, but the cell fluorescences that were basically unobservable before amplification can now be observed, but the cell outlines are still incomplete. When ethanol anhydrous enhancement is implemented (lower left), the cell fluorescence is significantly enhanced. After digital signal amplification, all cell outlines are completely presented and clearly visible.

[0084] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for visualizing the recognition profile of rare single cells, characterized in that, Comprising the following steps, S1: Performing cell membrane fluorescence staining for specifically recognizing the cell membrane markers of rare single cells to obtain cell solution I with water or aqueous solution as the solvent; S2: Adding a fluorescence enhancing reagent to the cell solution I to obtain cell solution II; the fluorescence enhancing reagent is an organic phase solution of a surfactant or a liquid organic alcohol, the surfactant is Tween-80, and the concentration of the surfactant is 1-16 mmol / L; S3: Performing microscopic imaging on the cell solution II; S4: Converting the photo of the microscopic imaging into a digital photo, and amplifying the digital signal in the digital photo to obtain a visualized cell contour map.

2. The method for visualizing the recognition profile of rare single cells according to claim 1, characterized in that, Before the step S1, an operation of fixing the rare single cells is further included.

3. The method for visualizing the recognition profile of rare single cells according to claim 1, characterized in that, Before the step S1, an operation of blocking the non-specific sites of the cell membrane of the rare single cells is further included.

4. The method for visualizing the recognition profile of rare single cells according to claim 1, characterized in that, In the step S2, the fluorescence enhancing reagent is added to the cell solution I directly or dropped onto the cell solution I on a carrier.

5. The method for visualizing the recognition profile of rare single cells according to claim 1, characterized in that, The liquid organic alcohol is selected from methanol, ethanol, propanol or isopropanol.

6. The method for visualizing the recognition profile of rare single cells according to claim 1, characterized in that, In the step S4, the photo of the microscopic imaging is converted into a digital photo by using a compiled program, and then the digital photo is subjected to specific digital signal amplification or non-specific digital signal amplification.

7. The method for visualizing the recognition profile of rare single cells according to claim 6, characterized in that, The non-specific digital signal amplification is to amplify all the digital signals in the photo, and the amplification multiple is based on the criterion that the matrix fluorescence cannot be observed, and at the same time, the cell contour is clearly visible.

8. The method for visualizing the recognition profile of rare single cells according to claim 6, characterized in that, The specific digital signal amplification is to amplify the signals with a signal-to-noise ratio greater than 3, and the amplification multiple is based on the criterion that the cell contour is clearly visible.

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