Fluorescence labeling reagent combination and application thereof

Abstract

The invention belongs to the technical field of fluorescence labeling, and particularly relates to a fluorescence labeling reagent combination and application thereof. The target spot of the fluorescence labeling reagent combination is polysaccharide, a sample containing the polysaccharide can be labeled, eukaryotic cells can be effectively labeled, seedless cells and prokaryotes can also be effectively labeled, the blank that the seedless cells and the prokaryotes cannot be labeled by a cell fluorescence labeling technology is filled, and the universality is higher; meanwhile, various fluorophores can be freely selected, different samples can be marked by different fluorophores, and fluorescence recognition is performed after mixing; in addition, by further limiting the molar ratio of all the components in the fluorescence labeling reagent combination, the efficiency of preparing the fluorescence labeling complex is improved, and cells are more easily labeled.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent labeling, and in particular relates to a fluorescent labeling reagent combination and application thereof. Background Art

[0002] Fluorescent labeling is a field that has attracted much attention in existing biolabeling technologies. With the development of biotechnology, fluorescent labeling has also spawned different application technologies, such as cell fluorescence counting, fluorescent cell imaging, etc.

[0003] The existing fluorescent labeling technology mainly labels cells by embedding fluorescent groups into the cell nucleus. However, this method has the following problems: 1) The fluorescent groups entering the cell may cause changes in the gene expression regulation and chromatin open state in the cell; 2) It is not suitable for anucleated cells, such as mature red blood cells of mammals, mature sieve tube cells of angiosperms, and prokaryotes; 3) Since the fluorescent marker needs to be embedded in the cell nucleus, there are only a few fixed fluorescent markers to choose from, such as acridine orange (AO), which leads to a lack of selectivity in the wavelength of the fluorescent marker. For example, AO (acridine orange, a small molecule dye, excitation at 488, emission at 525) in AO / PI fluorescent staining can penetrate normal cell membranes, label cell nucleic acids and emit green fluorescence, while PI (propidium iodide, a large molecule dye, excitation at 535, emission at 600) labels dead cell nucleic acids and emits red fluorescence. DAPI in DAPI fluorescent staining is 4',6-diamidino-2-phenylindole, a fluorescent dye that can penetrate intact cell membranes and strongly bind to DNA. DAPI can quickly enter living cells and bind to DNA, so DAPI is also considered a toxic substance and carcinogen for organisms.

[0004] Other fluorescent labeling methods, such as immunofluorescence labeling, can use multiple fluorescent labels to label specific proteins or peptides on the cell surface without acting on the cell nucleus. However, due to the specificity of their antibodies, they lack wide adaptability. One type of immunofluorescence labeling cannot be applied to multiple cell types, and the process is cumbersome and costly. Summary of the invention

[0005] The first aspect of the present invention aims to provide a fluorescent labeling reagent combination.

[0006] The purpose of the second aspect of the present invention is to provide the use of the fluorescent labeling reagent combination of the first aspect in the preparation of a fluorescent labeling complex.

[0007] The third aspect of the present invention aims to provide a method for preparing a fluorescently labeled complex.

[0008] The fourth aspect of the present invention aims to provide a fluorescently labeled complex.

[0009] The fifth aspect of the present invention aims to provide a fluorescent labeling kit.

[0010] The sixth aspect of the present invention aims to provide a sample labeling method.

[0011] The purpose of the seventh aspect of the present invention is to provide applications of the fluorescent labeling reagent combination of the first aspect, the preparation method of the third aspect, the fluorescent labeling complex of the fourth aspect, the fluorescent labeling kit of the fifth aspect, and / or the sample labeling method of the sixth aspect.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides a fluorescent labeling reagent combination, comprising: component A, component B and component C;

[0014] The A component comprises: avidin or its analogue;

[0015] The B component comprises:

[0016] a1) Concanavalin A or its analogs labeled with biotin or its analogs; and / or

[0017] a2) a fusion protein containing biotin or its analogs and concanavalin A or its analogs;

[0018] The C component comprises: a sample tag sequence, one end of which is connected to biotin or its analogue, and the other end of which is connected to a fluorescent group.

[0019] Preferably, the molar ratio of component A, component B and component C is 32:(0.5-13):(60-330).

[0020] Preferably, the avidin analog refers to a substance that has the same function as avidin and can bind to biotin or its analogs, such as streptavidin, neutravidin, and rhizoavidin.

[0021] Preferably, the concanavalin A analog refers to a substance that has the same function as concanavalin A and can bind to polysaccharides, such as other types of lectins (eg wheat germ agglutinin, peanut agglutinin, soybean agglutinin).

[0022] Preferably, the biotin analogue refers to a substance that has the same function as biotin and can bind to avidin or its analogues, such as Iminibiotin (iminobiotin), Desthiobiotin (desulfobiotin), etc.

[0023] Preferably, the fluorescent group can be any fluorescent group that can be modified on a single-stranded nucleotide; further preferably, the fluorescent group includes at least one of Cy3, Alexa488, FITC, Cy5, Rhodamine Red, JOE, FAM, TET, HEX, and ROX.

[0024] Preferably, the sample tag sequence comprises a tag sequence.

[0025] Preferably, the sample tag sequence further comprises a first primer binding sequence.

[0026] Preferably, the sample tag sequence further comprises a cell tag binding sequence.

[0027] Preferably, the cell tag binding sequence comprises a second primer binding sequence or a polyadenylation tail sequence.

[0028] Preferably, the fluorescent labeling reagent combination further comprises an instruction manual, which records the method of use, specifically:

[0029] b1) mixing component A with component B to obtain a mixture, and incubating;

[0030] b2) mixing the incubated mixture with component C and incubating to obtain a fluorescently labeled complex.

[0031] The second aspect of the present invention provides use of the fluorescent labeling reagent combination of the first aspect in preparing a fluorescent labeling complex.

[0032] The third aspect of the present invention provides a method for preparing a fluorescently labeled complex, comprising the step of using the fluorescently labeled reagent combination of the first aspect of the present invention.

[0033] Preferably, the preparation method comprises the following steps:

[0034] b1) mixing component A of the first aspect of the present invention with component B to obtain a mixture, and incubating;

[0035] b2) mixing the incubated mixture with component C of the first aspect of the present invention, and incubating to obtain a fluorescently labeled complex.

[0036] The fourth aspect of the present invention provides a fluorescent labeling complex, comprising: component A, component B and component C in the first aspect of the present invention;

[0037] The A component is connected to the B component;

[0038] The A component is connected to the C component;

[0039] The molar ratio of the A component, the B component and the C component is 32: (0.5-13): (60-330).

[0040] Preferably, the molar ratio of the component A, the component B and the component C is 32:(0.8-12.8):(64-320); further 32:6.4:128.

[0041] Preferably, a fluorescently labeled complex is obtained by the preparation method of the third aspect of the present invention.

[0042] A fifth aspect of the present invention provides a fluorescent labeling kit, comprising at least one of c1) to c2) and a buffer:

[0043] c1) the fluorescent labeling reagent combination according to the first aspect of the present invention;

[0044] c2) The fluorescently labeled complex according to the fourth aspect of the present invention.

[0045] Preferably, the buffer solution is a buffer solution containing metal ions, and the metal ions contain at least one of calcium ions, magnesium ions and manganese ions.

[0046] Preferably, the buffer is a commonly used buffer in the art; preferably, the buffer comprises at least one of Dulbecco's phosphate buffered saline (DPBS), phosphate buffered saline (PBS), and tris-HCl buffer (Tris-HCl).

[0047] A sixth aspect of the present invention provides a sample labeling method, comprising at least one of e1) to e4):

[0048] e1) a step of using the fluorescent labeling reagent combination according to the first aspect of the present invention;

[0049] e2) the preparation method according to the third aspect of the present invention;

[0050] e3) using the step of fluorescently labeling the complex according to the fourth aspect of the present invention;

[0051] e4) The steps of using the fluorescent labeling kit according to the fifth aspect of the present invention.

[0052] Preferably, the sample labeling method comprises the following steps: mixing a sample, a buffer solution, and the fluorescent labeling complex of the fourth aspect of the present invention, and incubating the obtained mixture to obtain a labeled sample.

[0053] Preferably, the buffer is the buffer in the fifth aspect of the present invention.

[0054] The seventh aspect of the present invention provides the use of the fluorescent labeling reagent combination of the first aspect of the present invention, the preparation method of the third aspect of the present invention, the fluorescent labeling complex of the fourth aspect of the present invention, the fluorescent labeling kit of the fifth aspect of the present invention, and / or the sample labeling method of the sixth aspect of the present invention.

[0055] Use of at least one of f1) to f5) in any of g1) to g4);

[0056] f1) The fluorescent labeling reagent combination according to the first aspect of the present invention;

[0057] f2) the preparation method according to the third aspect of the present invention;

[0058] f3) The fluorescently labeled complex according to the fourth aspect of the present invention;

[0059] f4) The fluorescent labeling kit according to the fifth aspect of the present invention;

[0060] f5) The sample labeling method according to the sixth aspect of the present invention;

[0061] g1) Sample labeling;

[0062] g2) sample imaging;

[0063] g3) sample counting;

[0064] g4) Single-cell sequencing.

[0065] Preferably, the sample is a sample comprising polysaccharides.

[0066] Preferably, the sample comprises at least one of eukaryotic cells, anucleate cells, and prokaryotes.

[0067] The beneficial effects of the present invention are:

[0068] The present invention provides a fluorescent labeling reagent combination, the target of which is polysaccharide, and the sample containing polysaccharide can be labeled: not only eukaryotic cells can be effectively labeled, but also anucleate cells and prokaryotes can be effectively labeled, filling the gap that cell fluorescence labeling technology cannot label anucleate cells and prokaryotes, and the universality is stronger; at the same time, a variety of fluorescent groups can be freely selected, and different samples can be labeled with different fluorescent groups, and then mixed for fluorescence identification; and the present invention improves the efficiency of preparing the fluorescent labeling complex by further limiting the molar ratio of each component in the fluorescent labeling reagent combination, and it is easier to label cells.

[0069] Furthermore, the present invention provides a method for preparing a fluorescently labeled complex. By further limiting the order of addition and the amount of each component in the fluorescently labeled reagent combination, the preparation efficiency of the fluorescently labeled complex is greatly improved, the content of invalid fluorescently labeled complexes is greatly reduced, and the carrying capacity of the sample label sequence of a single effective fluorescently labeled complex is significantly increased, making it easier to label cells.

[0070] Furthermore, the present invention provides a fluorescent labeling kit, which activates concanavalin A or its analogs in the fluorescent labeling complex by further adding a buffer for incubating the fluorescent labeling complex with the sample, especially a buffer containing at least one of calcium ions, magnesium ions and manganese ions, thereby greatly improving the binding efficiency and stability of the fluorescent labeling complex with the cell surface glycoprotein, increasing the number of connections of the fluorescent labeling complex on the cell surface, and making it easier to label cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Schematic diagram of fluorescent labeling on the cell surface.

[0072] Figure 2 These are images of cells labeled with the fluorescent labeling kit of Example 7: wherein A is a bright field image of cells labeled with the fluorescent labeling kit of Example 7; and B is a fluorescent image of cells labeled with the fluorescent labeling kit of Example 7.

[0073] Figure 3 These are images of cells labeled with the fluorescent labeling kit of Comparative Example 3: wherein A is a bright field image of cells labeled with the fluorescent labeling kit of Comparative Example 3; and B is a fluorescent image of cells labeled with the fluorescent labeling kit of Comparative Example 3.

[0074] Figure 4 These are images of cells labeled with the fluorescent labeling kit of Comparative Example 4: wherein A is a bright field image of cells labeled with the fluorescent labeling kit of Comparative Example 4; and B is a fluorescent image of cells labeled with the fluorescent labeling kit of Comparative Example 4.

[0075] Figure 5 These are images of cells labeled with the fluorescent labeling kit of Example 10: wherein A is a bright field image of cells labeled with the fluorescent labeling kit of Example 10; and B is a fluorescent image of cells labeled with the fluorescent labeling kit of Example 10.

[0076] Figure 6 These are images of cells labeled with the fluorescent labeling kit of Example 8: wherein A is a bright field image of cells labeled with the fluorescent labeling kit of Example 8; and B is a fluorescent image of cells labeled with the fluorescent labeling kit of Example 8.

[0077] Figure 7 These are images of cells labeled with the fluorescent labeling kit of Example 9: wherein A is a bright field image of cells labeled with the fluorescent labeling kit of Example 9; and B is a fluorescent image of cells labeled with the fluorescent labeling kit of Example 9.

[0078] Figure 8 These are cell images after PBMCs were labeled with the fluorescent labeling kit of Example 7: wherein A is a bright field image of cells after PBMCs were labeled with the fluorescent labeling kit of Example 7; and B is a fluorescent image of cells after PBMCs were labeled with the fluorescent labeling kit of Example 7.

[0079] Fig. 9 These are images of cells after the cell nuclei are labeled with the fluorescent labeling kit of Example 7: wherein A is a bright field image of cells after the cell nuclei are labeled with the fluorescent labeling kit of Example 7; and B is a fluorescent image of cells after the cell nuclei are labeled with the fluorescent labeling kit of Example 7. DETAILED DESCRIPTION

[0080] In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0081] The first aspect of the present invention provides a fluorescent labeling reagent combination, comprising: component A, component B and component C;

[0082] The A component comprises: avidin or its analogue;

[0083] The B component comprises:

[0084] a1) Concanavalin A or its analogs labeled with biotin or its analogs; and / or

[0085] a2) a fusion protein containing biotin or its analogs and concanavalin A or its analogs;

[0086] The C component comprises: a sample tag sequence, one end of which is connected to biotin or its analogue, and the other end of which is connected to a fluorescent group.

[0087] Preferably, the molar ratio of component A, component B and component C is 32:(0.5-13):(60-330); further 32:(0.8-12.8):(64-320); further 32:6.4:128.

[0088] Preferably, the A component, B component and C component exist independently and do not mix with each other.

[0089] Preferably, the avidin analog refers to a substance that has the same function as avidin and can bind to biotin or its analogs, such as streptavidin, neutravidin, and rhizoavidin.

[0090] Preferably, the concanavalin A analog refers to a substance that has the same function as concanavalin A and can bind to polysaccharides, such as other types of lectins (eg wheat germ agglutinin, peanut agglutinin, soybean agglutinin).

[0091] Preferably, the biotin analogue refers to a substance that has the same function as biotin and can bind to avidin or its analogues, such as Iminibiotin (iminobiotin), Desthiobiotin (desulfobiotin), etc.

[0092] Preferably, the A component comprises: avidin.

[0093] Preferably, the B component comprises:

[0094] a1) biotinylated concanavalin A; and / or

[0095] a2) A fusion protein containing biotin and concanavalin A.

[0096] Preferably, the B component comprises: biotin-labeled concanavalin A.

[0097] Preferably, the C component comprises: a sample tag sequence, one end of the sample tag sequence is connected to biotin, and the other end of the sample tag sequence is connected to a fluorescent group.

[0098] Preferably, the sample tag sequence comprises a tag sequence.

[0099] Preferably, the sample tag sequence further comprises a first primer binding sequence.

[0100] Preferably, the first primer binding sequence is used for subsequent PCR amplification of the sample tag sequence, and its length is generally between 16 and 28 bases. The design of the first primer binding sequence needs to follow the primer design principles.

[0101] Preferably, the first primer binding sequence is located at the 5' end or 3' end of the tag sequence; further located at the 5' end of the tag sequence.

[0102] Preferably, the sample label sequence also includes a cell label binding sequence, and the cell label binding sequence can be used for subsequent connection with the cell label in single-cell sequencing. The cell label is respectively connected to the sequencing material and the sample label sequence in the cell, and sequenced separately. Finally, based on the sequencing material and the sample label sequence with the same cell label, it is known which sample the sequencing material comes from.

[0103] Preferably, the cell tag binding sequence is located at the 5' end or 3' end of the tag sequence (ie, at the end not connected to the first primer binding sequence); further located at the 3' end of the tag sequence.

[0104] Preferably, the cell tag binding sequence comprises a second primer binding sequence or a polyadenylation tail sequence.

[0105] Preferably, the cell label binding sequence is a second primer binding sequence, and the second primer binding sequence also needs to follow the primer design principles. The second primer binding sequence is used for subsequent connection with the cell label in single-cell sequencing through base complementary pairing. In the process of single-cell sequencing, the cell label is respectively connected to the sequencing material and the sample label sequence in the cell, and sequenced separately. Finally, based on the sequencing material and the sample label sequence with the same cell label, it is known which sample the sequencing material comes from.

[0106] Preferably, the cell label binding sequence is a poly-A sequence tail, which is specifically used for mRNA sequencing. Since the cell label in single-cell mRNA sequencing carries a poly-T tail sequence, the sample label sequence can be combined with the cell label sequence, thereby sequencing the sequencing material with the cell label and the sample label sequence with the cell label respectively, and knowing which sample the sequencing material comes from based on the same cell label.

[0107] Preferably, the sample tag sequence is as shown in SEQ ID NO.1.

[0108] Preferably, a random sequence for increasing the length of the sample label sequence may be included between the first primer binding sequence and the label sequence and between the second primer binding sequence and the label sequence, thereby increasing the recovery efficiency of the sample label sequence in subsequent single-cell sequencing.

[0109] Preferably, the tag sequence is a specified sequence on some sequencing platforms, and each sequencing platform has its own set of tag sequences, such as a barcode sequence or an index sequence.

[0110] Preferably, the length of the tag sequence is 8 to 15 bp.

[0111] Preferably, the 5' end of the sample tag sequence is connected to biotin or its analogues, and the 3' end is connected to a fluorescent group.

[0112] Preferably, the fluorescent group can be any fluorescent group that can be modified on a single-stranded nucleotide; further preferably, the fluorescent group includes at least one of Cy3, Alexa488, FITC, Cy5, Rhodamine Red, JOE, FAM, TET, HEX, and ROX.

[0113] Preferably, the fluorescent labeling reagent combination is used for sample labeling, sample imaging, sample counting and / or single cell sequencing.

[0114] Preferably, the sample is a sample comprising polysaccharides.

[0115] Preferably, the sample comprises at least one of eukaryotic cells, anucleate cells, and prokaryotes.

[0116] Preferably, the fluorescent labeling reagent combination further comprises an instruction manual, which records the method of use, specifically:

[0117] b1) mixing component A with component B to obtain a mixture, and incubating;

[0118] b2) mixing the incubated mixture with component C and incubating to obtain a fluorescently labeled complex.

[0119] Preferably, the incubation conditions in b1) and b2) are incubation at 23-28°C for 10-20 min.

[0120] Preferably, the incubation in b2) is carried out under light-proof conditions.

[0121] The second aspect of the present invention provides use of the fluorescent labeling reagent combination of the first aspect in preparing a fluorescent labeling complex.

[0122] The third aspect of the present invention provides a method for preparing a fluorescently labeled complex, comprising the step of using the fluorescently labeled reagent combination of the first aspect of the present invention.

[0123] Preferably, the preparation method comprises the following steps:

[0124] b1) mixing component A of the first aspect of the present invention with component B to obtain a mixture, and incubating;

[0125] b2) mixing the incubated mixture with component C and incubating to obtain a fluorescently labeled complex.

[0126] Preferably, the incubation conditions in b1) and b2) are incubation at 23-28°C for 10-20 min.

[0127] Preferably, the incubation in b2) is carried out under light-proof conditions.

[0128] A fourth aspect of the present invention provides a fluorescent labeling complex comprising: component A, component B and component C;

[0129] The A component comprises: avidin or its analogue;

[0130] The B component comprises:

[0131] a1) Concanavalin A or its analogs labeled with biotin or its analogs; and / or

[0132] a2) a fusion protein containing biotin or its analogs and concanavalin A or its analogs;

[0133] The C component comprises: a sample tag sequence, one end of which is connected to biotin or its analogue, and the other end of which is connected to a fluorescent group;

[0134] The A component is connected to the B component;

[0135] The A component is connected to the C component;

[0136] The molar ratio of the A component, the B component and the C component is 32: (0.5-13): (60-330).

[0137] Preferably, the molar ratio of the component A, the component B and the component C is 32:(0.8-12.8):(64-320); further 32:6.4:128.

[0138] Preferably, the A component and the B component are linked via avidin or its analogue in the A component and biotin or its analogue in the B component.

[0139] Preferably, the A component and the C component are linked via avidin or its analogue in the A component and biotin or its analogue in the C component.

[0140] Preferably, the avidin analog refers to a substance that has the same function as avidin and can bind to biotin or its analogs, such as streptavidin, neutravidin, and rhizoavidin.

[0141] Preferably, the concanavalin A analog refers to a substance that has the same function as concanavalin A and can bind to polysaccharides, such as other types of lectins (eg wheat germ agglutinin, peanut agglutinin, soybean agglutinin).

[0142] Preferably, the biotin analogue refers to a substance that has the same function as biotin and can bind to avidin or its analogues, such as Iminibiotin (iminobiotin), Desthiobiotin (desthiobiotin, etc.).

[0143] Preferably, the A component comprises: avidin.

[0144] Preferably, the B component comprises:

[0145] a1) biotinylated concanavalin A; and / or

[0146] a2) A fusion protein containing biotin and concanavalin A.

[0147] Preferably, the B component comprises: biotin-labeled concanavalin A.

[0148] Preferably, the C component comprises: a sample tag sequence, one end of the sample tag sequence is connected to biotin, and the other end of the sample tag sequence is connected to a fluorescent group.

[0149] Preferably, the sample tag sequence comprises a tag sequence.

[0150] Preferably, the sample tag sequence further comprises a first primer binding sequence.

[0151] Preferably, the first primer binding sequence is used for subsequent PCR amplification of the sample tag sequence, and its length is generally between 16 and 28 bases. The design of the first primer binding sequence needs to follow the primer design principles.

[0152] Preferably, the first primer binding sequence is located at the 5' end or 3' end of the tag sequence; further located at the 5' end of the tag sequence.

[0153] Preferably, the sample label sequence also includes a cell label binding sequence, and the cell label binding sequence can be used for subsequent connection with the cell label in single-cell sequencing. The cell label is respectively connected to the sequencing material and the sample label sequence in the cell, and sequenced separately. Finally, based on the sequencing material and the sample label sequence with the same cell label, it is known which sample the sequencing material comes from.

[0154] Preferably, the cell tag binding sequence is located at the 5' end or 3' end of the tag sequence (ie, at the end not connected to the first primer binding sequence); further located at the 3' end of the tag sequence.

[0155] Preferably, the cell tag binding sequence comprises a second primer binding sequence or a polyadenylation tail sequence.

[0156] Preferably, the cell label binding sequence is a second primer binding sequence, and the second primer binding sequence also needs to follow the primer design principles. The second primer binding sequence is used for subsequent connection with the cell label in single-cell sequencing through base complementary pairing. In the process of single-cell sequencing, the cell label is respectively connected to the sequencing material and the sample label sequence in the cell, and sequenced separately. Finally, based on the sequencing material and the sample label sequence with the same cell label, it is known which sample the sequencing material comes from.

[0157] Preferably, the cell label binding sequence is a poly-A sequence tail, which is specifically used for mRNA sequencing. Since the cell label in single-cell mRNA sequencing carries a poly-T tail sequence, the sample label sequence can be combined with the cell label sequence, thereby sequencing the sequencing material with the cell label and the sample label sequence with the cell label respectively, and knowing which sample the sequencing material comes from based on the same cell label.

[0158] Preferably, the sample tag sequence is as shown in SEQ ID NO.1.

[0159] Preferably, a random sequence for increasing the length of the sample label sequence may be included between the first primer binding sequence and the label sequence and between the second primer binding sequence and the label sequence, thereby increasing the recovery efficiency of the sample label sequence in subsequent single-cell sequencing.

[0160] Preferably, the tag sequence is a specified sequence on some sequencing platforms, and each sequencing platform has its own set of tag sequences, such as a barcode sequence or an index sequence.

[0161] Preferably, the length of the tag sequence is 8 to 15 bp.

[0162] Preferably, the 5' end of the tag sequence is connected to biotin or its analogues, and the 3' end is connected to a fluorescent group.

[0163] Preferably, the fluorescent group can be any fluorescent group that can be modified on a single-stranded nucleotide; further preferably, the fluorescent group includes at least one of Cy3, Alexa488, FITC, Cy5, Rhodamine Red, JOE, FAM, TET, HEX, and ROX.

[0164] Preferably, a fluorescently labeled complex is obtained by the preparation method of the third aspect of the present invention.

[0165] Preferably, the fluorescent labeling complex is used for sample labeling, sample imaging, sample counting and / or single cell sequencing.

[0166] Preferably, the sample is a sample comprising polysaccharides.

[0167] Preferably, the sample comprises at least one of eukaryotic cells, anucleate cells, and prokaryotes.

[0168] A fifth aspect of the present invention provides a fluorescent labeling kit, comprising at least one of c1) to c2) and a buffer:

[0169] c1) the fluorescent labeling reagent combination according to the first aspect of the present invention;

[0170] c2) The fluorescently labeled complex according to the fourth aspect of the present invention.

[0171] Preferably, the buffer solution is a buffer solution containing metal ions, and the metal ions contain at least one of calcium ions, magnesium ions and manganese ions.

[0172] Preferably, the buffer is a commonly used buffer in the art; preferably, the buffer comprises at least one of Dulbecco's phosphate buffered saline (DPBS), phosphate buffered saline (PBS), and tris-HCl buffer (Tris-HCl).

[0173] Preferably, the metal ions include: at least one of magnesium ions and manganese ions, and calcium ions.

[0174] Preferably, the metal ions include magnesium ions and calcium ions.

[0175] Preferably, the molar ratio of the magnesium ions to the calcium ions is (40-60):90; further 49:90.

[0176] Preferably, the kit further comprises bovine serum albumin.

[0177] Preferably, the fluorescent labeling kit is used for sample labeling, sample imaging, sample counting and / or single-cell sequencing.

[0178] Preferably, the sample is a sample comprising polysaccharides.

[0179] Preferably, the sample comprises at least one of eukaryotic cells, anucleate cells, and prokaryotes.

[0180] A sixth aspect of the present invention provides a detection system, comprising at least one of d1) to d3) and a microscope:

[0181] d1) The fluorescent labeling reagent combination according to the first aspect of the present invention;

[0182] d2) The fluorescently labeled complex according to the fourth aspect of the present invention;

[0183] d3) The fluorescent labeling kit according to the fifth aspect of the present invention.

[0184] Preferably, the microscope comprises a fluorescence microscope.

[0185] Preferably, the detection system is used for sample labeling, sample imaging and / or sample counting.

[0186] Preferably, the sample is a sample comprising polysaccharides.

[0187] Preferably, the sample comprises at least one of eukaryotic cells, anucleate cells, and prokaryotes.

[0188] A seventh aspect of the present invention provides a sample labeling method, comprising at least one of e1) to e5):

[0189] e1) a step of using the fluorescent labeling reagent combination according to the first aspect of the present invention;

[0190] e2) the preparation method according to the third aspect of the present invention;

[0191] e3) using the step of fluorescently labeling the complex according to the fourth aspect of the present invention;

[0192] e4) using the step of the fluorescent labeling kit according to the fifth aspect of the present invention;

[0193] e5) The steps of adopting the detection system of the sixth aspect of the present invention.

[0194] Preferably, the step e5) adopts the fluorescent labeling reagent combination, fluorescent labeling complex, and / or fluorescent labeling kit in the detection system of the sixth aspect of the present invention.

[0195] Preferably, the sample labeling method comprises the following steps: mixing a sample, a buffer solution, and the fluorescent labeling complex of the fourth aspect of the present invention, and incubating the obtained mixture to obtain a labeled sample.

[0196] Preferably, the buffer solution is a buffer solution containing metal ions, and the metal ions contain at least one of calcium ions, magnesium ions and manganese ions.

[0197] Preferably, the metal ions include: at least one of magnesium ions and manganese ions, and calcium ions.

[0198] Preferably, the metal ions include magnesium ions and calcium ions.

[0199] Preferably, the final concentration of the metal ions in the mixture is 1.2-1.4 mM; further 1.2065-1.3463 mM.

[0200] Preferably, the final concentration of the magnesium ions in the mixture is 0.4-0.5 mM; further 0.4253-0.4746 mM.

[0201] Preferably, the final concentration of the manganese ions in the mixture is 0.4-0.5 mM; further 0.4253-0.4746 mM.

[0202] Preferably, the final concentration of the calcium ions in the mixture is 0.7-0.9 mM; further 0.7812-0.87170 mM.

[0203] Preferably, the buffer further comprises BSA.

[0204] Preferably, the buffer is a commonly used buffer in the art; preferably, the buffer comprises at least one of Dulbecco's phosphate buffered saline (DPBS), phosphate buffered saline (PBS), and tris-HCl buffer (Tris-HCl).

[0205] Preferably, the sample is a sample suspension, which is prepared by conventional methods in the art according to different samples.

[0206] Preferably, the sample is a sample comprising polysaccharides.

[0207] Preferably, the sample comprises at least one of eukaryotic cells, anucleate cells, and prokaryotes.

[0208] Preferably, the incubation condition is 23-28° C. for 10-20 min.

[0209] Preferably, the method further comprises the steps of: washing and resuspending.

[0210] The eighth aspect of the present invention provides the application of the fluorescent labeling reagent combination of the first aspect of the present invention, the preparation method of the third aspect of the present invention, the fluorescent labeling complex of the fourth aspect of the present invention, the fluorescent labeling kit of the fifth aspect of the present invention, the detection system of the sixth aspect of the present invention, and / or the sample labeling method of the seventh aspect of the present invention.

[0211] Use of at least one of f1) to f6) in any of g1) to g4);

[0212] f1) The fluorescent labeling reagent combination according to the first aspect of the present invention;

[0213] f2) the preparation method according to the third aspect of the present invention;

[0214] f3) The fluorescently labeled complex according to the fourth aspect of the present invention;

[0215] f4) The fluorescent labeling kit according to the fifth aspect of the present invention;

[0216] f5) The detection system of the sixth aspect of the present invention;

[0217] f6) The sample labeling method according to the seventh aspect of the present invention;

[0218] g1) Sample labeling;

[0219] g2) sample imaging;

[0220] g3) sample counting;

[0221] g4) Single-cell sequencing.

[0222] Preferably, the sample is a sample comprising polysaccharides.

[0223] Preferably, the sample comprises at least one of eukaryotic cells, anucleate cells, and prokaryotes.

[0224] The present invention is further described in detail below through specific examples.

[0225] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0226] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0227] In the following examples / comparative examples: biotin-labeled concanavalin A was purchased from Sigma-Aldrich, catalog number C2272; streptavidin was purchased from NEB, catalog number N7021S; the sample tag sequence (GCTGCGCTCGATGCAAAATAGAAACCCTBAAAAAAAAAAAAAAAAAAAAAAAAAAAA, SEQ ID NO.1) labeled with biotin at the 5' end and fluorescent group (FITC) at the 3' end was ordered from Bio-Tech; Dulbecco's phosphate buffer (DPBS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] purchased from Gibco, catalog number 14040133; bovine serum albumin (BSA) purchased from Bioscientific, catalog number C506031; Dulbecco's phosphate buffered saline (DPBS) (without Ca 2+ and Mg 2+ ) was purchased from Gibco, catalog number 14190144.

[0228] Example 1 A fluorescent labeling reagent combination

[0229] A fluorescent labeling reagent combination comprises: 4 μL of biotin-labeled concanavalin A with a concentration of 1.6 μM, 2 μL of streptavidin with a concentration of 16 μM, 1.28 μL of a sample label sequence with a 5' end labeled with biotin and a 3' end labeled with a fluorescent group with a concentration of 100 μM.

[0230] Example 2 A fluorescent labeling reagent combination

[0231] A fluorescent labeling reagent combination comprises: 8 μL of biotin-labeled concanavalin A with a concentration of 1.6 μM, 2 μL of streptavidin with a concentration of 16 μM, 3.2 μL of a sample label sequence with a 5' end labeled with biotin and a 3' end labeled with a fluorescent group with a concentration of 100 μM.

[0232] Example 3 A fluorescent labeling reagent combination

[0233] A fluorescent labeling reagent combination comprises: 0.5 μL of biotin-labeled concanavalin A with a concentration of 1.6 μM, 2 μL of streptavidin with a concentration of 16 μM, 0.64 μL of a sample label sequence with a 5' end labeled with biotin and a 3' end labeled with a fluorescent group with a concentration of 100 μM.

[0234] Example 4 A fluorescent labeled complex

[0235] A fluorescent labeling complex comprises the steps of using the reagent combination of Example 1, specifically as follows:

[0236] 1) Add 4 μL of biotin-labeled concanavalin A to 2 μL of streptavidin, vortex to mix, centrifuge the resulting mixture, and incubate at room temperature for 10 minutes;

[0237] 2) 1.28 μL of the sample tag sequence labeled with biotin at the 5' end and fluorescent group at the 3' end was added to the mixed solution of step 1) under light-proof conditions, vortexed to mix, centrifuged instantaneously, and incubated at room temperature in the dark for 10 minutes to obtain a fluorescently labeled complex.

[0238] Example 5 A fluorescent labeled complex

[0239] A fluorescent labeling complex comprises the steps of using the reagent combination of Example 2, specifically as follows:

[0240] 1) Add 8 μL of biotin-labeled concanavalin A to 2 μL of streptavidin, vortex to mix, centrifuge the resulting mixture, and incubate at room temperature for 10 minutes;

[0241] 2) Add 3.2 μL of the sample tag sequence labeled with biotin at the 5' end and fluorescent group at the 3' end to the mixed solution of step 1) under light-proof conditions, vortex mix, centrifuge instantaneously, and incubate at room temperature in the dark for 10 minutes to obtain a fluorescently labeled complex.

[0242] Example 6 A fluorescent labeled complex

[0243] A fluorescent labeling complex comprises the steps of using the reagent combination of Example 3, specifically as follows:

[0244] 1) Add 0.5 μL of biotin-labeled concanavalin A to 2 μL of streptavidin, vortex to mix, centrifuge the resulting mixture, and incubate at room temperature for 10 minutes;

[0245] 2) Add 0.64 μL of the sample tag sequence labeled with biotin at the 5' end and fluorescent group at the 3' end to the mixed solution of step 1) under light-proof conditions, vortex mix, centrifuge instantaneously, and incubate at room temperature in the dark for 10 minutes to obtain a fluorescently labeled complex.

[0246] Example 7 A fluorescent labeling kit

[0247] A fluorescent labeling kit, comprising: the fluorescent labeling complex of Example 4 and Dulbecco's phosphate buffer (DP BS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49mM)].

[0248] Example 8 A fluorescent labeling kit

[0249] A fluorescent labeling kit, comprising: the fluorescent labeling complex of Example 5 and Dulbecco's phosphate buffer (DP BS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49mM)].

[0250] Example 9 A fluorescent labeling kit

[0251] A fluorescent labeling kit, comprising: the fluorescent labeling complex of Example 6 and Dulbecco's phosphate buffer (DP BS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49mM)].

[0252] Example 10 A fluorescent labeling kit

[0253] A fluorescent labeling kit, comprising: the fluorescent labeling complex of Example 4 and Dulbecco's phosphate buffer (DP BS) (without Ca2+ and Mg 2+ ).

[0254] Comparative Example 1 A fluorescent labeling reagent combination

[0255] A fluorescent labeling reagent combination comprises: 4 μL of biotin-labeled concanavalin A with a concentration of 1.6 μM, 2 μL of streptavidin with a concentration of 16 μM, 1.28 μL of a sample label sequence with a 5' end labeled with biotin and a 3' end labeled with a fluorescent group with a concentration of 100 μM.

[0256] Comparative Example 2 A fluorescent labeled complex

[0257] A fluorescent labeling complex comprises the steps of using the fluorescent labeling reagent combination of Comparative Example 1, and the specific steps are as follows:

[0258] 1) Add 2 μL of streptavidin to 1.28 μL of the sample tag sequence labeled with biotin at the 5' end and fluorescent group at the 3' end under light-proof conditions, vortex to mix, centrifuge the resulting mixture, and incubate at room temperature in the dark for 10 minutes;

[0259] 2) Add 4 μL of biotin-labeled concanavalin A to the mixed solution of step 1) in the dark, vortex mix, centrifuge instantaneously, and incubate in the dark at room temperature for 10 minutes to obtain a fluorescently labeled complex.

[0260] Comparative Example 3: A fluorescent labeling kit

[0261] A fluorescent labeling kit, comprising: the fluorescent labeling complex of comparative example 2 and Dulbecco's phosphate buffer (DPBS) (without Ca 2+ and Mg 2+ ).

[0262] Comparative Example 4: A fluorescent labeling kit

[0263] A fluorescent labeling kit, comprising: the fluorescent labeling complex of comparative example 2 and Dulbecco's phosphate buffer (DP BS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49mM)].

[0264] Effect Example

[0265] 1. Cell imaging using the fluorescent labeling kits of Examples 7, 8, 9, 10 and Comparative Examples 3 and 4

[0266] (1) A method for cell imaging, comprising the steps of using the fluorescent labeling kit of Example 7, wherein the specific steps are as follows:

[0267] 1) Prepare a cell suspension of mammalian cell line 3T3 by conventional methods, and use Dulbecco's phosphate buffered saline (DPBS) [containing Ca2+] in the fluorescent labeling kit of Example 7 containing 1% BSA. 2+ (0.9 mM) and Mg 2+ (0.49 mM)] for two washes, and then washed with Dulbecco's phosphate buffered saline (DPBS) containing 1% BSA [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] to resuspend the cells to obtain a cell suspension with a density of 3860 cells / μL;

[0268] 2) Transfer 51.8 μL of cell suspension to a 1.5 mL centrifuge tube;

[0269] 3) The fluorescent labeling complex (4+2+1.28=7.28 μL) in the fluorescent labeling kit in Example 7 was added to the centrifuge tube in step 2), and the mixture was diluted with Dulbecco's phosphate buffer (DPBS) in the fluorescent labeling kit in Example 7 containing 1% BSA [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] Add the liquid volume in the centrifuge tube to 100 μL (Ca in the system 2 + Mg 2+ The final concentration was 0.8345mM, 0.4543mM), and the mixture was gently pipetted until completely mixed. The mixture was incubated at room temperature in the dark for 10 minutes. The schematic diagram of cell surface fluorescence labeling is shown in FIG. Figure 1 As shown;

[0270] 4) After the incubation, the cells were washed with Dulbecco's phosphate buffer (DPBS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] Supplement the suspension to 1 mL, wash the cell suspension by centrifugation (select 500 g centrifugation for 5 min), collect the cells to the bottom of the tube, and remove the supernatant;

[0271] 5) Repeat the washing once and remove the supernatant;

[0272] 6) Add 100 μL of Dulbecco's phosphate buffered saline (DPBS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)】Resuspend the cells and gently pipette until completely mixed;

[0273] 7) Observe the cell suspension obtained in step 6) through a fluorescence microscope with a corresponding fluorescence channel, and compare the cell positions under bright field and fluorescence conditions to confirm the status of cell fluorescence labeling.

[0274] (2) A method for cell imaging, comprising the steps of using the fluorescent labeling kit of Example 10, wherein the specific steps are as follows:

[0275] 1) A cell suspension of mammalian cell line 3T3 was prepared by conventional methods, and the suspension was diluted with Dulbecco's phosphate buffered saline (DPBS) (without Ca2+) in the fluorescent labeling kit of Example 10 containing 1% BSA. 2+ and Mg 2+ ) were washed twice, and then washed with Dulbecco's phosphate buffered saline (DPBS) (without Ca) in the fluorescent labeling kit of Example 10 containing 1% BSA. 2+ and Mg 2+ ) resuspend the cells to obtain a cell suspension with a density of 3860 cells / μL;

[0276] 2) Transfer 51.8 μL of cell suspension to a 1.5 mL centrifuge tube;

[0277] 3) The fluorescent labeled complex (4+2+1.28=7.28 μL) in the fluorescent labeling kit in Example 10 was added to the centrifuge tube in step 2), and the solution was washed with Dulbecco's phosphate buffer (DPBS) (without Ca2+) in the fluorescent labeling kit in Example 10 containing 1% BSA. 2+ and Mg 2+ ) Add the liquid volume in the centrifuge tube to 100 μL (Ca in the system 2+ Mg 2+ The final concentration was 0.8345 mM and 0.4543 mM), and the mixture was gently pipetted until completely mixed, and incubated at room temperature in the dark for 10 minutes;

[0278] 4) After the incubation, the cells were washed with Dulbecco's phosphate buffered saline (DPBS) (without Ca) in the fluorescent labeling kit of Example 10 containing 1% BSA. 2+ and Mg 2+ ) Replenish the suspension to 1 mL, wash the cell suspension by centrifugation (select 500 g for 5 min), collect the cells to the bottom of the tube, and remove the supernatant;

[0279] 5) Repeat the washing once and remove the supernatant;

[0280] 6) Add 100 μL of Dulbecco's phosphate buffered saline (DPBS) (without Ca) in the fluorescent labeling kit of Example 10 containing 1% BSA. 2+ and Mg2+ ) Resuspend the cells and gently pipette until completely mixed;

[0281] 7) Observe the cell suspension obtained in step 6) through a fluorescence microscope with a corresponding fluorescence channel, and compare the cell positions under bright field and fluorescence conditions to confirm the status of cell fluorescence labeling.

[0282] (3) A method for cell imaging, comprising the steps of using the fluorescent labeling kit of Comparative Example 3, wherein the specific steps are as follows:

[0283] 1) A cell suspension of mammalian cell line 3T3 was prepared by conventional methods, and the suspension was diluted with Dulbecco's phosphate buffered saline (DPBS) (without Ca2+) in the fluorescent labeling kit of Comparative Example 3 containing 1% BSA. 2+ and Mg 2+ ) were washed twice, and then washed with Dulbecco's phosphate buffer (DPBS) (without Ca) in the fluorescent labeling kit of Comparative Example 3 containing 1% BSA. 2+ and Mg 2 + ) resuspend the cells to obtain a cell suspension with a density of 3860 cells / μL;

[0284] 2) Transfer 51.8 μL of cell suspension to a 1.5 mL centrifuge tube;

[0285] 3) The fluorescent labeled complex (2+1.28+4=7.28 μL) in the fluorescent labeling kit in comparative example 3 was added to the centrifuge tube in step 2), and the Dulbecco's phosphate buffer (DPBS) (without Ca) in the fluorescent labeling kit in comparative example 3 containing 1% BSA was used. 2+ and Mg 2+ ) Add the liquid volume in the centrifuge tube to 100 μL (Ca in the system 2+ Mg 2+ The final concentration was 0.8345 mM and 0.4543 mM), and the mixture was gently pipetted until completely mixed, and incubated at room temperature in the dark for 10 minutes;

[0286] 4) After the incubation, the Dulbecco's phosphate buffer (DPBS) (without Ca) in the fluorescent labeling kit of Comparative Example 3 containing 1% BSA was used. 2+ and Mg 2+ ) Replenish the suspension to 1 mL, wash the cell suspension by centrifugation (select 500 g for 5 min), collect the cells to the bottom of the tube, and remove the supernatant;

[0287] 5) Repeat the washing once and remove the supernatant;

[0288] 6) Add 100 μL of Dulbecco's phosphate buffered saline (DPBS) (without Ca) in the fluorescent labeling kit of Comparative Example 3 containing 1% BSA 2+ and Mg 2+ ) Resuspend the cells and gently pipette until completely mixed;

[0289] 7) Observe the cell suspension obtained in step 6) through a fluorescence microscope with a corresponding fluorescence channel, and compare the cell positions under bright field and fluorescence conditions to confirm the status of cell fluorescence labeling.

[0290] (4) A method for cell imaging, comprising the steps of using the fluorescent labeling kit of Comparative Example 4, wherein the specific steps are as follows:

[0291] 1) A cell suspension of mammalian cell line 3T3 was prepared by conventional methods, and the suspension was diluted with Dulbecco's phosphate buffer (DPBS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] for two washes, and then washed with Dulbecco's phosphate buffer (DPBS) [containing Ca (0.49 mM)] in the fluorescent labeling kit of Comparative Example 4 containing 1% BSA 2+ (0.9 mM) and Mg 2+ (0.49 mM)] to resuspend the cells to obtain a cell suspension with a density of 3860 cells / μL;

[0292] 2) Transfer 51.8 μL of cell suspension to a 1.5 mL centrifuge tube;

[0293] 3) Add the fluorescent labeled complex (2+1.28+4=7.28 μL) in the fluorescent labeling kit in comparative example 4 to the centrifuge tube in step 2), and dilute with Dulbecco's phosphate buffer (DPBS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] Add the liquid volume in the centrifuge tube to 100 μL (Ca in the system 2 + Mg 2+ The final concentration was 0.8345 mM and 0.4543 mM), and the mixture was gently pipetted until completely mixed, and incubated at room temperature in the dark for 10 minutes;

[0294] 4) After the incubation, the Dulbecco's phosphate buffer (DPBS) (containing Ca 2+ and Mg 2+) Replenish the suspension to 1 mL, wash the cell suspension by centrifugation (select 500 g for 5 min), collect the cells to the bottom of the tube, and remove the supernatant;

[0295] 5) Repeat the washing once and remove the supernatant;

[0296] 6) Add 100 μL of Dulbecco's phosphate buffer (DPBS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)】Resuspend the cells and gently pipette until completely mixed;

[0297] 7) Observe the cell suspension obtained in step 6) through a fluorescence microscope with a corresponding fluorescence channel, and compare the cell positions under bright field and fluorescence conditions to confirm the status of cell fluorescence labeling.

[0298] (5) A method for cell imaging, comprising the steps of using the fluorescent labeling kit of Example 8, wherein the specific steps are as follows:

[0299] 1) Prepare a cell suspension of mammalian cell line 3T3 by conventional methods, and use Dulbecco's phosphate buffered saline (DPBS) [containing Ca2+] in the fluorescent labeling kit of Example 8 containing 1% BSA. 2+ (0.9 mM) and Mg 2+ (0.49 mM)] for two washes, and then washed with Dulbecco's phosphate buffered saline (DPBS) containing 1% BSA [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] to resuspend the cells to obtain a cell suspension with a density of 3860 cells / μL;

[0300] 2) Transfer 51.8 μL of cell suspension to a 1.5 mL centrifuge tube;

[0301] 3) The fluorescent labeled complex (8+2+3.2=13.2 μL) in the fluorescent labeling kit in Example 8 was added to the centrifuge tube in step 2) and the mixture was diluted with Dulbecco's phosphate buffer (DPBS) in the fluorescent labeling kit in Example 8 containing 1% BSA [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] Add the liquid volume in the centrifuge tube to 100 μL (Ca in the system 2 + Mg 2+ The final concentration was 0.7812 mM, 0.4253 mM), and the mixture was gently pipetted until completely mixed, and incubated at room temperature in the dark for 10 minutes;

[0302] 4) After the incubation, the cells were washed with Dulbecco's phosphate buffer (DPBS) containing 1% BSA in the fluorescent labeling kit of Example 8 [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] Supplement the suspension to 1 mL, wash the cell suspension by centrifugation (select 500 g centrifugation for 5 min), collect the cells to the bottom of the tube, and remove the supernatant;

[0303] 5) Repeat the washing once and remove the supernatant;

[0304] 6) Add 100 μL of Dulbecco's phosphate buffered saline (DPBS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)】Resuspend the cells and gently pipette until completely mixed;

[0305] 7) Observe the cell suspension obtained in step 6) through a fluorescence microscope with a corresponding fluorescence channel, and compare the cell positions under bright field and fluorescence conditions to confirm the status of cell fluorescence labeling.

[0306] (6) A method for cell imaging, comprising the steps of using the fluorescent labeling kit of Example 9, wherein the specific steps are as follows:

[0307] 1) Prepare a cell suspension of mammalian cell line 3T3 by conventional methods, and use Dulbecco's phosphate buffered saline (DPBS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] for two washes, and then washed with Dulbecco's phosphate buffered saline (DPBS) containing 1% BSA [containing Ca2 + (0.9 mM) and Mg2 + (0.49 mM)] to resuspend the cells to obtain a cell suspension with a density of 3860 cells / μL;

[0308] 2) Transfer 51.8 μL of cell suspension to a 1.5 mL centrifuge tube;

[0309] 3) The fluorescent labeling complex (0.5+2+0.64=3.14 μL) in the fluorescent labeling kit in Example 9 was added to the centrifuge tube in step 2), and the mixture was diluted with Dulbecco's phosphate buffer (DPBS) in the fluorescent labeling kit in Example 9 containing 1% BSA [containing Ca 2+ (0.9 mM) and Mg 2+(0.49 mM)] Add the liquid volume in the centrifuge tube to 100 μL (Ca in the system 2+ Mg 2+ The final concentration was 0.8717 mM and 0.4746 mM), and the mixture was gently pipetted until completely mixed, and incubated at room temperature in the dark for 10 minutes;

[0310] 4) After the incubation, the cells were washed with Dulbecco's phosphate buffer (DPBS) containing 1% BSA in the fluorescent labeling kit of Example 9 [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)] Supplement the suspension to 1 mL, wash the cell suspension by centrifugation (select 500 g centrifugation for 5 min), collect the cells to the bottom of the tube, and remove the supernatant;

[0311] 5) Repeat the washing once and remove the supernatant;

[0312] 6) Add 100 μL of Dulbecco's phosphate buffered saline (DPBS) [containing Ca 2+ (0.9 mM) and Mg 2+ (0.49 mM)】Resuspend the cells and gently pipette until completely mixed;

[0313] 7) Observe the cell suspension obtained in step 6) through a fluorescence microscope with a corresponding fluorescence channel, and compare the cell positions under bright field and fluorescence conditions to confirm the status of cell fluorescence labeling.

[0314] (7) A method for cell imaging, comprising the steps of using the fluorescent labeling kit of Example 7, the steps are the same as (1), the only difference being that PBMC is used.

[0315] (8) A method for cell imaging, comprising the steps of using the fluorescent labeling kit of Example 7, the steps are the same as (1), the only difference being that a cell nucleus is used.

[0316] The cell imaging images using the fluorescent labeling kit of Example 7 and the fluorescent labeling kit of Comparative Example 4 are as follows: Figure 2 , 4As shown in the figure, fluorescence can be effectively observed by using the fluorescent labeling kit of Example 7, while effective fluorescence can hardly be observed by using the fluorescent labeling kit of Comparative Example 4. This is because the fluorescent labeling complex in the fluorescent labeling kit of Comparative Example 4 first incubates streptavidin and the sample label sequence labeled with biotin at the 5' end and the fluorescent group at the 3' end at a molar ratio of 1:4, and then adds 0.2 times of biotin-labeled concanavalin A to form a fluorescent labeling complex. After the sample label sequence binds to streptavidin, a lot of concanavalin A cannot bind to streptavidin, resulting in the ineffective preparation of the fluorescent labeling complex. The rate is low, and the fluorescent labeling of a single cell cannot be effectively observed; the fluorescent labeling complex in the fluorescent labeling kit of Example 7 is obtained by the following method: first, biotin-labeled concanavalin A and streptavidin are incubated at a molar ratio of 1:5 to allow the concanavalin A tetramer to fully interact with streptavidin, and then a sample label sequence with 5'-end labeled biotin and 3'-end labeled fluorescent group, which is 4 times the amount of streptavidin, is added for incubation, thereby increasing the efficiency of preparing the fluorescent labeling complex, greatly reducing the content of invalid fluorescent labeling complexes, and significantly increasing the carrying capacity of the sample label sequence of a single effective fluorescent labeling complex.

[0317] The cell imaging images using the fluorescent labeling kit of Example 7 and the fluorescent labeling kit of Example 10 are as follows: Figure 2 , 5 As shown in the figure, fluorescence can be effectively observed using the fluorescent labeling kits of Example 7 and Example 10, but the fluorescence of the fluorescent labeling kit of Example 10 is less. This is because when the fluorescent labeling kit of Example 10 is used, the concanavalin A in the fluorescent labeling complex is not activated, resulting in low efficiency of the fluorescent labeling complex binding to the sugar chain of the cell surface glycoprotein, and it is easy to fall off from the cell surface, resulting in a decrease in the number of fluorescent labeling complexes marked on the cell surface. When the fluorescent labeling kit of Example 7 is used to incubate the fluorescent labeling complex and cells, Ca is added. 2+ and Mg 2+ , activating concanavalin A in the fluorescent labeling complex, thereby greatly improving the binding efficiency and stability of the fluorescent labeling complex with the sugar chain of the cell surface glycoprotein, and increasing the number of connections of the fluorescent labeling complex on the cell surface.

[0318] The cell imaging images using the fluorescent labeling kit of Example 7 and the fluorescent labeling kit of Comparative Example 3 are as follows: Figure 2 , 3As shown: almost no effective fluorescence can be observed using the fluorescent labeling kit of comparative example 3, while fluorescence can be effectively observed using the fluorescent labeling kit of embodiment 7; this is because most of the fluorescent labeling complexes in the fluorescent labeling kit of comparative example 3 are invalid fluorescent labeling complexes (incomplete structure, not carrying concanavalin A), and the concanavalin A in the effective fluorescent labeling complexes is not activated, specifically as follows: the fluorescent labeling complexes in the fluorescent labeling kit of comparative example 3 are first incubated with streptavidin and a sample label sequence labeled with biotin at the 5' end and a fluorescent group at the 3' end at a molar ratio of 1:4, and then 0.2 times the amount of biotin-labeled concanavalin A of streptavidin is added for incubation to form a fluorescent labeling complex. After the sample label sequence binds to streptavidin, a lot of concanavalin A cannot bind to streptavidin, resulting in low preparation efficiency of the fluorescent labeling complex. At the same time, due to The concanavalin A in the fluorescently labeled complex is not activated, resulting in low efficiency of binding of the fluorescently labeled complex to the sugar chain of the cell surface glycoprotein, and easy detachment from the cell surface, resulting in a decrease in the number of fluorescently labeled complexes labeled on the cell surface, and the fluorescent label cannot be effectively observed; and the fluorescently labeled complex in the fluorescent labeling kit of Example 7 is obtained by the following method: first, biotin-labeled concanavalin A and streptavidin are incubated at a molar ratio of 1:5, so that the concanavalin A tetramer fully interacts with streptavidin, and then a sample label sequence with a 5' end labeled with biotin and a 3' end labeled with a fluorescent group that is 4 times the amount of streptavidin is added for incubation, thereby making the preparation efficiency of the fluorescently labeled complex high, greatly reducing the content of invalid fluorescently labeled complexes, and significantly increasing the carrying capacity of the sample label sequence of a single effective fluorescently labeled complex, and when incubating the fluorescently labeled complex and cells, Ca is added 2+ and Mg 2+ , activating concanavalin A in the fluorescent labeling complex, thereby greatly improving the binding efficiency and stability of the fluorescent labeling complex with the sugar chain of the cell surface glycoprotein, and increasing the number of connections of the fluorescent labeling complex on the cell surface, so that the fluorescent label can be clearly and effectively observed.

[0319] The cell imaging images using the fluorescent labeling kit of Example 8 and the fluorescent labeling kit of Example 9 are as follows: Figure 6 , 7 As shown, the cell imaging effect using the fluorescent labeling kit of Example 8 and the fluorescent labeling kit of Example 9 is equivalent to the cell imaging effect using the fluorescent labeling kit of Example 7.

[0320] The cell imaging diagram of PBMC and cell nucleus labeled by the fluorescent labeling kit of Example 7 is as follows: Figure 8 , 9As shown: the cell imaging of PBMC and cell nucleus labeled with the fluorescent labeling kit of Example 7 is equivalent to the cell imaging effect of 3T3 labeled with the fluorescent labeling kit of Example 7.

[0321] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A fluorescent labeling reagent combination, comprising: component A, component B and component C; The A component comprises: avidin or its analogue; The B component comprises: a1) Concanavalin A or its analogs labeled with biotin or its analogs; and / or a2) a fusion protein containing biotin or its analogs and concanavalin A or its analogs; The C component comprises: a sample tag sequence, one end of which is connected to biotin or its analogue, and the other end of which is connected to a fluorescent group.

2. The fluorescent labeling reagent combination according to claim 1, characterized in that: The molar ratio of component A, component B and component C is 32: (0.5-13): (60-330); Preferably, the avidin analog refers to a substance having the same function as avidin and capable of binding to biotin or its analog; further comprising at least one of streptavidin, neutravidin, and rhizoavidin; Preferably, the concanavalin A analog refers to a substance having the same function as concanavalin A and capable of binding to polysaccharides; further comprising other types of lectins; further comprising: at least one of wheat germ lectin, peanut lectin, and soybean lectin; Preferably, the biotin analogue refers to a substance having the same function as biotin and capable of binding to avidin or its analogue; further comprising at least one of iminobiotin and desthiobiotin; Preferably, the fluorescent group can be any fluorescent group that can be modified on a single-stranded nucleotide; further preferably, the fluorescent group includes at least one of Cy3, Alexa488, FITC, Cy5, Rhodamine Red, JOE, FAM, TET, HEX, and ROX; Preferably, the sample tag sequence comprises a tag sequence; Preferably, the sample tag sequence further comprises a first primer binding sequence; Preferably, the sample tag sequence further comprises a cell tag binding sequence; Preferably, the cell tag binding sequence comprises a second primer binding sequence or a polyadenylic acid tail sequence; Preferably, the fluorescent labeling reagent combination further comprises instructions, which record the method of use, specifically: b1) mixing component A with component B to obtain a mixture, and incubating; b2) mixing the incubated mixture with component C and incubating to obtain a fluorescently labeled complex.

3. A method for preparing a fluorescently labeled complex, comprising: the steps of using the fluorescently labeled reagent combination according to claim 1 or 2; Preferably, the preparation method comprises the following steps: b1) mixing the component A of claim 1 or 2 with the component B to obtain a mixture, and incubating; b2) mixing the incubated mixture with the component C of claim 1 or 2, and incubating to obtain a fluorescently labeled complex.

4. A fluorescent labeling complex, comprising: component A, component B and component C as described in claim 1 or 2; The A component is connected to the B component; The A component is connected to the C component; The molar ratio of the A component, the B component and the C component is 32: (0.5-13): (60-330).

5. The fluorescent labeling complex according to claim 4, characterized in that: The molar ratio of component A, component B and component C is 32: (0.8-12.8): (64-320); Preferably, the fluorescently labeled complex is prepared by the preparation method described in claim 3.

6. A fluorescent labeling kit, comprising at least one of c1) to c2) and a buffer: c1) The fluorescent labeling reagent combination according to claim 1 or 2; c2) The fluorescently labeled complex according to claim 4 or 5.

7. The fluorescent labeling kit according to claim 6, characterized in that: The buffer is a buffer containing metal ions, and the metal ions include: at least one of calcium ions, magnesium ions and manganese ions; Preferably, the buffer comprises at least one of Dulbecco's phosphate buffer, phosphate buffer and Tris-HCl buffer.

8. A sample labeling method, comprising at least one of e1) to e4): e1) using the fluorescent labeling reagent combination according to claim 1 or 2; e2) The preparation method according to claim 3; e3) using the fluorescent labeling complex as described in claim 4 or 5; e4) The step of using the fluorescent labeling kit according to claim 6 or 7.

9. The method according to claim 8, characterized in that The sample labeling method comprises the following steps: mixing a sample, a buffer, and the fluorescent labeling complex according to claim 4 or 5, and incubating the obtained mixture to obtain a labeled sample; Preferably, the buffer is the buffer described in claim 7.

10. Use of at least one of f1) to f5) in any of g1) to g4); f1) The fluorescent labeling reagent combination according to claim 1 or 2; f2) The preparation method according to claim 3; f3) The fluorescently labeled complex according to claim 4 or 5; f4) The fluorescent labeling kit according to claim 6 or 7; f5) The sample labeling method according to claim 8 or 9; g1) Sample labeling; g2) sample imaging; g3) sample counting; g4) Single-cell sequencing; Preferably, the sample is a sample containing polysaccharides; Preferably, the sample comprises at least one of eukaryotic cells, anucleate cells, and prokaryotes.

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