Fluorescent dye for blood cell analysis and blood cell dyeing method

Abstract

The invention provides a fluorescent dye for blood cell analysis. The fluorescent dye has the following structural general formula I, wherein X is C (CH3) 2, O or S; m is an integer of 1-6; r1 and R2 are respectively and independently selected from H, C1-10 alkyl, C1-6 alkyl-OR7, halogen or benzyl, and R7 is H or C1-8 alkyl; r3 is NR5R6, NHR9, OH or halogen; wherein R5 and R6 are H, C1-2 alkyl groups or C1-C6 alkyl groups OR8, and R5 and R6 are not C2 alkyl groups at the same time; r9 is a C1-6 alkyl group OR8; r8 is H or a C1-8 alkyl group; r4 is a C1-10 alkyl group; y <-> is negative ions.

Description

Technical Field

[0001] This invention relates to a new class of fluorescent dyes in the field of fine chemicals, and in particular to a fluorescent dye for blood cell analysis and a method for staining blood cells. Background Technology

[0002] Reticulocytes are present in normal whole blood samples. Reticulocytes are nearly mature red blood cells and contain a large amount of nucleic acid. They are an important indicator of the red blood cell production status in the body and are often used to monitor anemia, blood metabolism, and hematopoietic capacity after massive blood loss.

[0003] Existing blood cell analyzers can detect reticulocytes. The detection principle is that reagents and fluorescent dyes act on blood cells in combination. The nucleic acid content in reticulocytes is significantly higher than that in mature red blood cells. The fluorescent dye binds to the nucleic acid substances in reticulocytes, generating a specific fluorescent signal under laser irradiation. The optical information is collected by a signal detector, and the proportion of reticulocytes is further calculated based on this optical information.

[0004] However, the existing Sysmex dyes are not specific enough for nucleic acids, resulting in high fluorescence in ordinary red blood cells but low fluorescence in reticulocytes containing nucleic acids. The signal-to-noise ratio is not good enough, especially since many reticulocytes that are about to mature and have low nucleic acid content cannot be effectively detected, leading to lower test results and failing to obtain more accurate results, which are lower than the results of manual microscopic examination. Summary of the Invention

[0005] To address at least one of the problems existing in the prior art, the present invention provides a fluorescent dye for blood cell analysis and a method for staining blood cells. The fluorescent dye has good nucleic acid specificity and can accurately identify reticulocytes.

[0006] This invention provides a fluorescent dye for blood cell analysis, the fluorescent dye having the following general structural formula I:

[0007]

[0008] in:

[0009] X is C(CH3)2, O, or S;

[0010] m is an integer from 1 to 6;

[0011] R1 and R2 are each independently selected from H, C1-10 alkyl, C1-6 alkyl-OR7, halogen or benzyl, and R7 is H or C1-8 alkyl;

[0012] R3 is NR5R6, NHR9, OH, or a halogen; wherein R5 and R6 are H, C1-2 alkyl, or C1-C6 alkyl OR8, and R5 and R6 are not both C2 alkyl; R9 is C1-6 alkyl OR8; R8 is H or C1-8 alkyl;

[0013] R4 is a C1-10 alkyl group;

[0014] Y - It is a negative ion.

[0015] In some implementations, R1 and R2 are each independently selected from H, C1-10 alkyl or benzyl.

[0016] In some embodiments, R4 is a C1-10 alkyl or benzyl group.

[0017] In some embodiments, Y is a halide ion.

[0018] In some embodiments, R7 is H or a C1-6 alkyl group.

[0019] This invention also provides a method for staining blood cells, the method comprising contacting the blood cells with the fluorescent dye described in any of the above embodiments.

[0020] When the compounds provided in the embodiments of the present invention are used as fluorescent dyes, they have the following beneficial effects:

[0021] 1. The fluorescent dye in the embodiments of the present invention is also referred to as a new compound in the present invention. A nitrogen-containing substituent is introduced into the molecule of the new compound, which increases the fluorescence quantum yield after the dye binds to nucleic acid and improves the detection sensitivity.

[0022] 2. The introduction of nitrogen-containing substituents into the molecule of this new compound appropriately increases molecular polarity and reduces the binding force on the hydrophobic regions inside molecules such as membrane lipids and proteins, thus exhibiting specific binding to nucleic acids.

[0023] 3. This new compound can use inexpensive, small-sized, and stable red semiconductor lasers as a light source, greatly reducing the cost of use. Attached Figure Description

[0024] Figure 1 This is a scatter plot of cell classification from Example 1 of the present invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity.

[0025] Figure 2 This is a scatter plot of cell classification from Example 2 of the present invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity.

[0026] Figure 3This is a scatter plot of cell classification in Example 3 of the present invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity. Detailed Implementation

[0027] Unless otherwise stated, the terms used in this invention have the following meanings.

[0028] The term "alkyl" as used in this invention includes both straight-chain alkyl and branched-chain alkyl. When referring to a single alkyl group such as "propyl," it specifically refers to a straight-chain alkyl group; when referring to a single branched-chain alkyl group such as "isopropyl," it specifically refers to a branched-chain alkyl group. For example, "C1-6 alkyl" includes C1-4 alkyl, C1-3 alkyl, methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Similar rules apply to other groups used in this specification.

[0029] The term "halogen" as used in this invention includes fluorine, chlorine, bromine, and iodine.

[0030] As used in this invention, the term "benzyl" refers to the -CH2-Ph group. When benzyl is modified with "optional substitution," it means that the benzyl group may exist in an unsubstituted form or may be substituted by a suitable substituent at any suitable position. Suitable substituents include, but are not limited to, H, C1-18 alkyl, CN, COOH, NH2, NO2, OH, SH, C1-6 alkoxy, C1-6 alkylamino, C1-6 amide, halogen, or C1-6 haloalkyl, as long as the final compound has the properties desired by this invention.

[0031] This invention provides a fluorescent dye for blood cell analysis, the fluorescent dye having the following general structural formula I:

[0032]

[0033] Wherein: X is C(CH3)2, O, or S; m is an integer from 1 to 6; R1 and R2 are each independently selected from H, C1-10 alkyl, C1-6 alkyl-OR7, halogen, or benzyl, wherein R7 is H or C1-8 alkyl; R3 is NR5R6, NHR9, OH, or halogen, wherein R5 and R6 are H, C1-2 alkyl, or C1-C6 alkyl-OR8, and R5 and R6 are not simultaneously C2 alkyl; R9 is C1-6 alkyl-OR8; R8 is H or C1-8 alkyl; R4 is C1-10 alkyl. Y - It is a negative ion, which can be any suitable negative ion, including but not limited to inorganic or organic negative ions, such as halide ions, ClO. 4- PF 6- BF 4- CH3COO - or OTs -The benzyl group may be optionally substituted with a substituent selected from the following: H, C1-18 alkyl, CN, COOH, NH2, NO2, OH, SH, C1-6 alkoxy, C1-6 alkylamino, C1-6 acylamino, halogen, or C1-6 haloalkyl; further, the benzyl group may be optionally substituted with COOH, NH2, OH, C1-6 alkoxy, or halogen.

[0034] In some embodiments, R1 and R2 are each independently selected from H, C1-10 alkyl or benzyl.

[0035] In some embodiments, R4 is a C1-10 alkyl or benzyl group.

[0036] In some embodiments, Y is a halide ion.

[0037] In some embodiments, R7 is H or a C1-6 alkyl group.

[0038] This invention also provides a method for staining blood cells, the method comprising contacting the blood cells with the fluorescent dye described in any of the above embodiments. The term "contact" as used in the embodiments may include contact in a solution or a solid phase.

[0039] When the compounds provided in the embodiments of the present invention are used as fluorescent dyes, they have the following beneficial effects:

[0040] 1. The introduction of nitrogen-containing substituents into the new compound molecule increases the fluorescence quantum yield after the dye binds to nucleic acid, thereby improving the detection sensitivity.

[0041] 2. The introduction of nitrogen-containing substituents into the new compound molecule appropriately increases the molecular polarity, reduces the binding force on the hydrophobic regions inside molecules such as membrane lipids and proteins, and exhibits specific binding to nucleic acids.

[0042] 3. The new compound can use inexpensive, small-sized, and stable red semiconductor lasers as a light source, greatly reducing the cost of use.

[0043] Example 1

[0044] The composition of Example 1 of the present invention was prepared using the following components:

[0045]

[0046] The structure of the fluorescent dye is as follows:

[0047]

[0048] Take 4 μL of blood sample and mix it with 1 mL of the above reagent to form a cell suspension, and incubate at 45°C for 5 seconds. Use laser flow cytometry (excitation source is a semiconductor laser, excitation wavelength is 635 nm) to detect nucleated red blood cells in the blood sample, detecting forward scattering (FS) information from 1 to 10 degrees and side fluorescence (SFL) information from 90 degrees. Generate a cell suspension based on the forward scattering and side fluorescence information. Figure 1 The scatter plot of cell classification shown indicates that the percentage of reticulocytes is 5.14%. In the same blood sample, confirmed by the new methylene blue staining method recommended by the International Committee for Standardization in Hematology (ICSH), the percentage of reticulocytes is 5.20%.

[0049] Example 2

[0050] The composition of Example 2 of the present invention was prepared using the following components:

[0051]

[0052] The structure of the fluorescent dye is as follows:

[0053]

[0054] Take 4 μL of blood sample and mix it with 1 mL of the above reagent to form a cell suspension, and incubate at 45°C for 8 seconds. Use laser flow cytometry (excitation source is a semiconductor laser, excitation wavelength is 635 nm) to detect nucleated red blood cells in the blood sample, detecting forward scatter (FS) information from 1 to 10 degrees and side fluorescence (SFL) information from 90 degrees. Generate a cell suspension based on the forward scatter and side fluorescence information. Figure 2 The scatter plot of cell classification shown indicates that the percentage of reticulocytes is 2.43%. In the same blood sample, confirmed by the new methylene blue staining method recommended by the International Committee for Standardization in Hematology (ICSH), the percentage of reticulocytes was 2.40%.

[0055] Example 3

[0056] The composition of Example 3 of the present invention was prepared using the following components:

[0057]

[0058] The structure of the fluorescent dye is as follows:

[0059]

[0060] Take 4 μL of blood sample and mix it with 1 mL of the above reagent to form a cell suspension, and incubate at 45°C for 10 seconds. Use laser flow cytometry (excitation source is a semiconductor laser, excitation wavelength is 635 nm) to detect nucleated red blood cells in the blood sample, detecting forward scattering (FS) information from 1 to 10 degrees and side fluorescence (SFL) information from 90 degrees. Generate a cell suspension based on the forward scattering and side fluorescence information. Figure 3 The scatter plot of cell classification shown indicates that the percentage of reticulocytes is 8.87%. In the same blood sample, confirmed by the new methylene blue staining method recommended by the International Committee for Standardization in Hematology (ICSH), the percentage of reticulocytes was 8.80%.

[0061] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered within the scope of protection of the present invention. Using the compound as a fluorescent dye is one application of the novel compound of the present invention; it should not be construed that the compound of the present invention is only used for fluorescent dyes. For those skilled in the art, based on the same mechanism of action of the compound of the present invention as a fluorescent dye, several simple inferences can be made to derive other applications of the compound of the present invention, and all such applications should be considered within the scope of protection of the present invention.

Claims

1. A fluorescent dye for blood cell analysis, characterized in that, The fluorescent dye has the following general structural formula I: in: X is C(CH3)2, O, or S; m is an integer from 1 to 6; R1 and R2 are each independently selected from H, C1-10 alkyl, C1-6 alkyl-OR7, halogen or benzyl, and R7 is H or C1-8 alkyl; R3 is NR5R6, NHR9, OH, or a halogen; wherein R5 and R6 are H, C1-2 alkyl, or C1-C6 alkyl OR8, and R5 and R6 are not both C2 alkyl; R9 is C1-6 alkyl OR8; R8 is H or C1-8 alkyl; R4 is a C1-10 alkyl group; Y - It is a negative ion.

2. The fluorescent dye for blood cell analysis as described in claim 1, characterized in that, R1 and R2 are each independently selected from H, C1-10 alkyl or benzyl.

3. The fluorescent dye for blood cell analysis according to claim 1, characterized in that, R4 is a C1-10 alkyl or benzyl group.

4. The fluorescent dye for blood cell analysis according to claim 1, characterized in that, The Y is a halide ion.

5. The fluorescent dye for blood cell analysis according to claim 1, characterized in that, R7 is H or C1-6 alkyl.

6. A method for staining blood cells, characterized in that, The method includes contacting blood cells with the fluorescent dye of any one of claims 1-5.

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