WO2024017247A1

WO2024017247A1 - Cyanine dye and preparation method therefor and use thereof, sample analysis method, and analyzer

Info

Publication number: WO2024017247A1
Application number: PCT/CN2023/107915
Authority: WO
Inventors: 刘宗俊; 陈庚文
Original assignee: 深圳迈瑞生物医疗电子股份有限公司
Priority date: 2022-07-20
Filing date: 2023-07-18
Publication date: 2024-01-25

Abstract

The present application relates to a fluorescent dye, and in particular, to a cyanine dye, and a preparation method therefor and a use thereof. Specifically, provided is a compound having a structure as represented by general formula I, or a hydrate, solvate, stereoisomer, tautomer, or crystal form thereof, wherein R1, R2 and R3 are as defined in the description. The provided cyanine dye has the advantages of good stability, good biological penetration and the like.

Description

Cyanine dye, its preparation method, use, sample analysis method and analyzer

Technical field

The present application relates to a fluorescent dye and its application, in particular to cyanine dye, its preparation method and its use, a sample analysis method and a sample analyzer. The present application also relates to conjugates comprising said cyanine dyes and compositions for staining biological samples.

Background technique

DNA (DeoxyriboNucleic Acid, deoxyribonucleic acid) is a type of biological macromolecule that carries genetic information. Normal cells of organisms generally have relatively stable DNA diploid content, but when pathological changes occur, abnormal changes occur; and the DNA content of different types of organisms generally differs. Therefore, the specific identification and accurate measurement of DNA, especially in living cells, are of great significance. The use of fluorescent dyes for qualitative or quantitative analysis of DNA has aroused the interest of scientific researchers due to its advantages of high sensitivity and fast response.

At present, commercial dyes mainly include phenanthridines, acridines, imidazoles and cyanine family types. However, these dye application processes have certain limitations. First, a larger portion of the dye exhibits fluorescence quenching after binding to DNA, resulting in low practical value in visualization applications such as fluorescence imaging. Second, most current dyes have poor specificity and cannot specifically bind to DNA, which greatly limits their application potential. Third, most current dyes themselves are highly toxic and carcinogenic, and biological samples need to be labeled by increasing the permeability of the cell membrane. However, this permeabilization treatment causes damage to cells and biological tissues, limiting its application in living cells.

Among many fluorescent dyes, cyanine fluorescent dyes have been widely used as biomolecule fluorescent probes due to their wide wavelength range, large molar extinction coefficient, and moderate fluorescence quantum yield. Although some cyanine dyes have been commercialized, most of these dyes have large molecules, complex structures, and low permeability to living cells. In addition, the Stoke shift of most dyes is small, resulting in severe crosstalk between the excitation spectrum and the emission spectrum, causing background interference and fluorescence self-quenching, which limits their applications.

Microorganisms are tiny organisms that exist in nature with small size, simple structure, and invisible to the naked eye. They can only be observed after magnification with the help of special instruments. Microorganisms include prokaryotes (such as bacteria, actinomycetes, mycoplasmas, rickettsiae, chlamydia, and spirochetes), eukaryotes (such as fungi, protozoa, algae), and non-cellular species (viruses and prions).

Many diseases are related to microorganisms. Related diseases caused by pathogenic microorganisms include diseases caused by bacteria, viruses, fungi and protozoa. Pathogenic microorganisms can cause blood infectious diseases, including bacteremia, sepsis, toxemia, sepsis, etc. Among them, sepsis is a systemic infection syndrome (SIRS) that can cause septic shock and multiple organ dysfunction, with a mortality rate as high as 30-50%. It can be seen that rapid detection of microorganisms is of great significance.

Currently, the gold standard for clinical diagnosis of microorganisms is blood culture. However, for blood culture, the entire process from blood to plate culture to result is cumbersome and time-consuming (usually more than 3-5 days), and the patient's condition is often very serious by the time the result comes out. Based on this, various new and effective detection methods have emerged one after another. These detection methods include immunoassays and electrochemical detection methods.

Immunoassays refer to the detection of specific microorganisms using specific reactions of antigens and antibodies, including traditional agglutinin tests and precipitation tests, as well as new enzyme-linked immunosorbent assays (ELISA), radioactive labeling methods, and chemiluminescence immunoassays. wait. Immunoassays are very specific and faster than traditional culture methods. However, due to shortcomings such as difficulty in obtaining antibodies, high antibody cost, insufficient detection sensitivity, and reactivity easily affected by the environment, immune detection methods are difficult to be widely used in clinical microbial detection.

The electrochemical detection method mainly uses the changes in electrical signals generated by the metabolic process of microorganisms to detect microorganisms. Common methods include impedance analysis, potential analysis, current analysis, etc. Equipment based on electrochemical detection methods is small and low-cost, but lacks sensitivity.

Blood cell parasitic microorganisms refer to parasites that live in the blood or blood cells of animals, such as malaria or Toxoplasma gondii. Parasites in the blood have a shorter onset period and are more harmful, so early and accurate diagnosis of parasites in blood cells is necessary for effective disease management and monitoring.

Currently, there are three main methods that can detect microorganisms parasitic in blood cells. The first method is the accurate detection and quantification of blood cell-parasitic microorganisms through microscopic examination of blood smears, however this is highly dependent on the training and skills of the operator. The second method is rapid diagnostic testing (RDT) based on antigen-antibody reactions, which does not require high operational skills but is generally expensive and lacks sufficient sensitivity for low-level blood cell parasites. The third method is to detect parasites in blood cells when using a hematology analyzer for routine blood screening. This method is simple, easy to operate, low-cost and does not rely on the training and skills of the operator.

However, the current accuracy of using blood cell analyzers to detect parasites in blood is still low.

Contents of the invention

In order to solve the above problems, the inventor of the present application obtained a new benzothiazole-based cyanine dye through molecular modification. Its thermal stability is significantly improved, and compared with the existing benzothiazole-based cyanine dye, Dye, the cyanine dye of the present invention has high biological penetration and is more suitable for cell dye and imaging.

compound

In a first aspect, the application provides a compound having a structure as shown in general formula I or a hydrate, solvate, stereoisomer, tautomer or crystalline form thereof,

in,

R ₁ and R ₂ are the same or different, and are independently selected from C _1-18 linear or branched alkyl, C _1-18 linear or branched alkylene-M, M is selected from sulfonic acid group, phenyl , carboxyl, thiol, amino;

R ₃ is selected from hydrogen, sulfonate group, halogen, cyano group, C _1-6 alkyl group, hydroxyl group, C _1-6 alkoxy group, and halogenated C _1-6 alkyl group;

Y does not exist or is a counter anion;

And limited to:

When R ₃ is hydrogen, R ₁ and R ₂ are not both methyl and R ₁ and R ₂ are not both benzyl.

In the compound of the present invention, R ₁ and R ₂ may be the same or different. In some embodiments, R ₁ and R ₂ are independently selected from C _1-6 linear alkyl, C _1-6 linear alkylene-M, and M is selected from sulfonate, phenyl, carboxyl, mercapto, Amino.

In some embodiments, at least one of R ₁ and R ₂ is a C _1-18 linear or branched alkylene-sulfonate group.

In some embodiments, R ₁ and R ₂ are different and independently selected from C _1-6 linear alkyl, benzyl, C _1-6 linear alkylene-carboxy, C _1-6 linear alkylene -Sulfonic acid group, C _1-6 linear alkylene-mercapto group, C _1-6 linear alkylene-amino group.

In some embodiments, R ₁ and R ₂ are the same and selected from C _1-6 linear alkyl, C _1-6 linear alkylene-sulfonate, C _1-6 linear alkylene-carboxy.

In some embodiments, _R3 is selected from hydrogen, sulfonate, halogen, cyano, C _1-6 alkyl.

In some embodiments, Y in general formula I is a counter anion. In this case, Y can be selected from halide ions (such as F ^- , Cl ^- , Br ^- , I ^- ), ClO ₄ ^- , PF ₆ ^- , CF ₃ SO ₃ ^- , BF ₄ ^- , acetate, methanesulfonate or p-toluenesulfonate.

In some embodiments, Y in Formula I is absent, in which case the compound may be an internal salt. "Internal salts" are also known in the art as "zwitterions". In some embodiments, the compounds of the present invention may contain both an acidic group (such as a sulfonic acid group or a carboxyl group) and a basic group (such as an amino or thiazole ring) within the molecule, the acidic group and the basic group Neutralize each other to form internal salts.

It should be understood that when a compound molecule contains multiple acidic groups and/or multiple basic groups, each acidic group and/or each basic group may serve as a salt-forming group. Salts formed by various salt formation methods of the compounds are included in the scope of the present invention.

In some embodiments, at least one of R ₁ and R ₂ is selected from C _1-18 linear or branched alkylene-sulfonate, C _1-18 linear or branched alkylene-carboxy.

The compounds of the present invention may have any of the following structures:

In some embodiments, a compound of the invention is a compound represented by Structural Formula 5, 6 or 9 above.

Conjugates and compositions for staining biological samples

The compounds of the present invention and their hydrates, solvates, stereoisomers, tautomers or crystal forms can be directly used for staining biological samples, or can also be used in the form of derivatives, and the derivatives include but are not limited to conjugate.

"Conjugate" as used herein refers to a compound formed by connecting a compound of the present invention, its hydrate, solvate, stereoisomer, tautomer or crystalline form to other molecules through a covalent bond. Molecules that can be conjugated to the compounds of the invention, their hydrates, solvates, stereoisomers, tautomers or crystalline forms can be molecules that specifically bind to cells or cellular components, including but not limited to antibodies, Antigens, receptors, ligands, enzymes, substrates, coenzymes, etc. Typically, the test sample is incubated with a fluorescent conjugate for a period of time so that the fluorescent conjugate specifically binds to certain cells or cellular components in the test sample. The binding of the fluorescent conjugate to cells or cellular components can also be determined by called dyeing. This staining step can be performed multiple times in sequence, or multiple stainings can be performed simultaneously with multiple conjugates. After the staining is completed, the sample is analyzed in an analytical instrument including an excitation light source that excites the fluorescent dye of the present invention in the conjugate and a measurement device that measures the emitted light generated by the excited fluorescent dye.

The present application also provides a composition for staining biological samples, wherein the composition comprises the compound of the present invention, its hydrate, solvate, stereoisomer, tautomer or crystal form, or the compound of the present invention. Inventive conjugates. In some embodiments, the biological sample is nucleic acid. In some embodiments, the biological sample is DNA.

use

The application also provides the use of the compounds of the invention, their hydrates, solvates, stereoisomers, tautomers or crystal forms, or the conjugates of the invention, or the compositions of the invention in biological samples. Use in dyeing. In some practical In embodiments, the biological sample is nucleic acid. In some embodiments, the biological sample is DNA.

The application also provides the compounds of the present invention or their hydrates, solvates, stereoisomers, tautomers or crystal forms, or the conjugates of the present invention, or the compositions of the present invention when using flow cytometry. Use of the technique to identify parasites in blood samples to be tested. In some embodiments, the parasite is selected from the group consisting of: roundworms, hookworms, tapeworms, Trichomonas vaginalis, liver flukes, Paragonimus westermanii, Toxoplasma gondii, cysticercosis suis, Trichinella spiralis, amoeba , Leishmania donovani, Plasmodium, Schistosoma, filarial worms, hydatid, scabies mites, hair follicle mites, lice, fleas.

The application also provides the compounds of the present invention or their hydrates, solvates, stereoisomers, tautomers or crystal forms, or the conjugates of the present invention, or the compositions of the present invention when using flow cytometry. The technology is used to identify microorganisms in blood samples to be tested or body fluid samples to be tested.

In some embodiments, the microorganism is selected from:

Bacteria (e.g., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Pertussis pertussis, Diphtheriae, Neisseria meningitidis, Mycobacterium tuberculosis, Clostridium tetani, Leprae, Group A hemolytic chain cocci, Brucella, Bacillus cholerae, Bacillus typhi, Bacillus anthracis, Neisseria gonorrhoeae, Vibrio cholerae, Pseudomonas klebsiella, and Salmonella paratyphi A, B or C),

Viruses (such as influenza virus, mumps virus, rubella virus, Japanese encephalitis virus, dengue virus, epidemic hemorrhagic fever virus, rabies virus, human papillomavirus, poliovirus, measles virus, varicella-zoster virus, hepatitis viruses, novel enterovirus 70, coxsackievirus A24 variant, human immunodeficiency virus, poxviruses (e.g. monkeypox virus)),

Fungi (such as Candida albicans, Trichophyton rubrum, Epidermophyton floccosum),

Mycoplasma (e.g. Mycoplasma pneumoniae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium),

Chlamydia (e.g. Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia domestica),

Rickettsiae (e.g., Rickettsia prowazekii, Rickettsia morgoni, Rickettsia rickettsiae, Rickettsia scrub typhus),

Actinomycetes (e.g. Actinomyces israelensis),

Spirochetes (e.g. Leptospira, Treponema pallidum).

In some embodiments, blood or body fluid refers to blood or body fluid from a mammal, especially a human. Among them, body fluid can be divided into urine, sweat, cerebrospinal fluid, serous cavity fluid, synovial fluid, etc. according to different parts. A small amount of the above-mentioned body fluids exists in the normal human body. The effusion in the serosal cavity includes pleural effusion, ascites and pericardial effusion, and the effusion in the joint cavity is synovial fluid (joint effusion).

General synthesis method

The compounds of the present invention can be synthesized by general methods in the art. Exemplarily, the benzothiazole compounds of the present invention can be synthesized by the following method: first starting from unsubstituted or substituted methylbenzothiazole, and heating it with R ₂ X (X is F, Cl, Br or I) The reaction was carried out under reflux to obtain intermediate I in the form of quaternary ammonium salt. Subsequently, the connecting molecule 4-hydroxyisophthalaldehyde and the intermediate I are heated and refluxed (the molar ratio of the intermediate I to 4-hydroxyisophthalaldehyde is greater than or equal to 2), so that the intermediate I and the connecting molecule Condensation gives the benzothiazole compound of the present invention.

When R ₂ is a C _1-18 linear or branched alkylene-sulfonic acid group, intermediate I can also be synthesized by the following method: starting from unsubstituted or substituted methylbenzothiazole and other raw materials, and making it with The appropriate sultone undergoes a ring-opening reaction to obtain intermediate I.

The above method is suitable for the case where R ₁ and R ₂ are the same.

When R ₁ and R ₂ are different, the benzothiazole compounds of the present invention can be synthesized by the following exemplary method: combining R ₁ or R ₂ substituted methylbenzothiazole with the connecting molecule 4-hydroxyisophthalaldehyde Heating and refluxing reaction (the molar ratio of 4-hydroxyisophthalaldehyde and methylbenzothiazole substituted by R ₁ or R ₂ is about 2) to obtain formyl-containing intermediate I'; make the obtained intermediate I' and R ₂ or R ₁ substituted methylbenzothiazole undergoes a condensation reaction to obtain the benzothiazole compound of the present invention.

In the above method, each intermediate or product can be recovered through separation and purification techniques known in the art to achieve the required purity.

Various raw materials used in the above method are commercially available, or can be prepared from raw materials known in the art by methods known to those skilled in the art or methods disclosed in the prior art.

Definition of Terms

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the experimental procedures used in this article are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the term "C _1-18 linear or branched alkyl" refers to a group obtained by removing one hydrogen atom from a linear or branched alkane containing 1 to 18 carbon atoms, specific examples thereof Including but not limited to: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, tert-butyl, isobutyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl base.

As used herein, "C _1-6 alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched chain alkane containing 1-6 carbon atoms. Specific examples thereof include but are not limited to: base, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, tert-butyl, isobutyl, etc.

As used herein, "C _1-6 linear alkyl" refers to a group obtained by removing one hydrogen atom from a linear alkane containing 1-6 carbon atoms. Specific examples thereof include but are not limited to: methyl , ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl.

C _1-18 straight-chain or branched alkylene refers to a group obtained by removing two hydrogen atoms from a straight-chain or branched alkane containing 1-18 carbon atoms. Specific examples include but are not limited to methylene, Ethylene, propylene, butylene, etc. The term "C _1-6 linear alkylene" refers to a group obtained by removing two hydrogen atoms from a linear alkane containing 1 to 6 carbon atoms.

As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

As used herein, the term "halogenated" means that a hydrogen on a group or compound is replaced by one or more halogen atoms, including fully halogenated and partially halogenated.

As used herein, the term "C _1-6 alkoxy" refers to a group formed in the manner of C 1-6 alkyl-O-.

As used herein, the term "solvate" (or "solvate") refers to a substance formed by the molecular association of a compound with an organic solvent (eg, methanol, ethanol, propanol, acetonitrile, etc.).

As used herein, the term "hydrate" refers to a substance formed by the association of a compound with water molecules.

As used herein, the term "crystalline form" refers to the crystal structure of a substance. During the crystallization of substances, due to the influence of various factors, the bonding methods within or between molecules change, resulting in different arrangements of molecules or atoms in the crystal lattice space, forming different crystal structures. The compound of the present invention can exist in one crystal structure or in multiple crystal structures, that is, it has "polymorphic form". The compounds of the invention may exist in different crystalline forms.

As used herein, the term "stereoisomer" includes conformational isomers and configurational isomers, wherein said conformational isomers Type isomers mainly include cis-trans isomers and optical isomers. The compounds of the present invention may exist in stereoisomeric forms and thus encompass all possible stereoisomeric forms, any combination or any mixture thereof. For example, a single enantiomer, a single diastereomer or a mixture of the above. When a compound of the present invention contains an olefinic double bond, it includes both cis and trans isomers, as well as any combination thereof, unless otherwise stated.

The compounds described in this invention may exist as tautomeric forms, which have different points of attachment of hydrogens through the displacement of one or more double bonds. For example, a ketone and its enol form are keto-enol tautomers. It is to be understood that the present invention encompasses all keto-enol tautomers of the compounds. Each tautomer and mixtures thereof are included within the scope of the invention.

Beneficial effects of the invention

Compared with the existing technology, the fluorescent dye of the present invention has one or more of the following beneficial effects:

1. Compared with existing dyes, the thermal stability of the dye of the present invention is significantly improved;

2. Dyes used for cell staining require high cell penetration. Compared with existing dyes, the dye of the present invention has high biological penetration and is more suitable for cell dyes and imaging;

3. The dye of the present invention can specifically bind to DNA, which is beneficial to the specific recognition and accurate measurement of DNA;

4. The dye of the present invention has good permeability to living cells, can enter cells to stain nucleic acids without damaging the cell membrane, and has low toxicity and low carcinogenicity;

5. The excitation light of the dye of the present invention is blue-green light with a smaller wavelength, which can identify tiny particles and improve the detection ability of small particles;

6. The dye of the present invention can use ordinary green semiconductor lasers as light sources, which greatly reduces the cost of use;

7. The dye of the present invention has a simple structure, the raw materials for its preparation are easily available, the synthesis yield is high, and it is easy to realize industrialization.

In order to solve the above problems, another task of the present invention is to provide a sample analysis method and sample analyzer that can accurately, sensitively, cost-effectively and quickly identify based on flow cytometry using fluorescent labeling technology. Microorganisms in blood or body fluids.

To this end, the second aspect of the present invention proposes a sample analysis method, which includes the following steps:

Using a dye reagent containing the first dye and a hemolytic agent for lysing red blood cells to process the same biological sample to be tested to obtain a sample liquid to be tested, wherein the biological sample to be tested is a blood sample to be tested or a body fluid sample to be tested, The first dye can dye microorganisms;

Make the particles in the sample liquid to be tested pass through the optical detection area one by one and irradiate the particles flowing through the optical detection area with light to obtain the scattered light information generated by the particles in the sample liquid to be tested after being irradiated with light. Fluorescence information including first fluorescence information from the first dye; and

Microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

In some embodiments, identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes:

Generate a first scatter plot based on the scattered light information and the first fluorescence information; and

Microorganisms in the sample fluid to be tested are identified based on the first scatter plot.

In some embodiments, the scattered light information includes forward scattered light information; and

Generating a first scatter plot based on the scattered light information and the first fluorescence information includes generating the first scatter plot based on the forward scattered light information and the first fluorescence information.

A microbial characteristic area and a leukocyte area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the microbial characteristic area is greater than the intensity of the first fluorescence information of the leukocyte area; and

Microorganisms in the sample fluid to be tested are identified based on the microorganism characteristic areas.

The number of microorganisms in the sample liquid to be tested is obtained based on the scattered light information and the first fluorescence information.

In some embodiments, the biological sample to be tested is a blood sample to be tested, and the sample analysis method further includes:

Parasites, especially Plasmodium, in the sample fluid to be tested are identified based on the scattered light information and the first fluorescence information.

In some embodiments, the biological sample to be tested is a blood sample to be tested, the dye reagent further includes a second dye different from the first dye, and the fluorescence information further includes second fluorescence information from the second dye, Wherein, the second dye can stain white blood cells in the blood; and

The sample analysis method further includes: obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

In some embodiments, the scattered light information includes side scattered light information and forward scattered light information;

Identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes: identifying microorganisms in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information. ;and

Obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information includes: obtaining the leukocyte classification results of the sample liquid to be tested based on the side scattered light information and the second fluorescence information. Classification results.

In some embodiments, the biological sample to be tested is a blood sample to be tested, the dye reagent further includes a second dye different from the first dye, and the fluorescence information further includes second fluorescence information from the second dye, Wherein, the second dye can stain white blood cells and nucleated red blood cells in the blood; and

The sample analysis method further includes: obtaining at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information.

In some embodiments, the scattered light information includes forward scattered light information;

Obtaining at least one of the white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information includes: based on the forward scattered light information and The second fluorescence information obtains at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample liquid to be tested.

In some embodiments, illuminating the particles flowing through the optical detection zone with light includes illuminating the particles flowing through the optical detection zone with light of a single wavelength.

In some embodiments, the first dye is a dye that specifically binds to DNA.

In some embodiments, the first dye includes a compound having the structure of Formula I above, wherein R ₁ and R ₂ are the same or different, and are independently selected from C _1-18 linear or branched alkyl, C _1-18 linear or branched alkylene-M, M is selected from sulfonate group, phenyl, carboxyl, mercapto, amino group, R ₃ is selected from hydrogen, sulfonate group, halogen, cyano group, C _1-6 Alkyl group, hydroxyl group, C _1-6 alkoxy group, halogenated C _1-6 alkyl group, Y does not exist or is a counter anion.

For more details and advantages of this compound, please refer to the above description and will not be repeated here.

A third aspect of the present invention proposes a sample analyzer, including:

A sampling device used to quantitatively absorb biological samples to be tested, where the biological samples to be tested are blood samples to be tested or body fluid samples to be tested;

A sample preparation device having a reaction tank and a reagent supply part, wherein the reaction tank is used to receive the biological sample to be tested drawn by the sampling device, and to receive a dye containing a first dye provided by the reagent supply part Reagents and hemolytic reagents for lysing red blood cells. The biological sample to be tested absorbed by the sampling device is mixed with the dye reagent and hemolytic reagent provided by the reagent supply part in the reaction tank to prepare a sample to be tested. Liquid, wherein the first dye can dye microorganisms;

Optical detection device, including a light source, a flow chamber, a scattered light detector and a fluorescence detector. The light source is used to emit a light beam to illuminate the flow chamber. The flow chamber is connected to the reaction cell and the sample liquid to be tested is The particles in the flow chamber can pass through the flow chamber one by one, the scattered light detector is used to detect the scattered light information generated by the particles passing through the flow chamber after being irradiated by light, and the fluorescence detector is used to detect the particles passing through the flow chamber. Fluorescence information generated by the particles after being illuminated by light, the fluorescence information including first fluorescence information from the first dye; and

A processor configured to acquire the scattered light information and the fluorescence information from the optical detection device, and identify microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information.

In some embodiments, the processor is further configured to perform the following steps when identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information:

The processor is further configured to generate the first scatter plot based on the forward scattered light information and the first fluorescence information.

In some embodiments, the processor is further configured to:

In some embodiments, the biological sample to be tested is a blood sample to be tested, and the processor is further configured to:

Parasites, especially Plasmodium, in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

In some embodiments, the biological sample to be tested is a blood sample to be tested, and the dye reagent further includes a second dye different from the first dye, and the second dye can stain white blood cells in the blood, and the The fluorescence information also includes second fluorescence information from the second dye; and

The processor is further configured to: obtain a leukocyte classification result of the sample fluid to be tested based on the scattered light information and the second fluorescence information.

In some embodiments, the scattered light information includes side scattered light information and forward scattered light information; and

The processor is further configured to: identify the test target based on the forward scattered light information and the first fluorescence information. microorganisms in the sample liquid, and obtain the leukocyte classification result of the sample liquid to be tested based on the side scattered light information and the second fluorescence information.

In some embodiments, the biological sample to be tested is a blood sample to be tested, and the dye reagent further includes a second dye that is different from the first dye, and the second dye can detect white blood cells and nucleated red blood cells in the blood. staining, the fluorescence information further comprising second fluorescence information from a second dye; and

The processor is further configured to: obtain at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information.

The processor is further configured to: identify microorganisms in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information, and identify microorganisms in the sample liquid to be tested based on the forward scattered light information and the second fluorescence information. The information obtains at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested.

In some embodiments, the light source is configured to illuminate the flow chamber with a single wavelength of light.

In some embodiments, the first dye is a dye that specifically binds to DNA.

In the sample analysis method proposed in the second aspect of the present invention and the sample analyzer proposed in the third aspect, first, a dye capable of staining microorganisms and a hemolytic agent used to dissolve red blood cells are used to process the blood sample to be tested or the body fluid sample to be tested, To obtain the sample liquid to be tested, and then use flow cytometry to obtain the scattered light information and fluorescence information of the sample liquid to be tested. Based on the scattered light information and the fluorescence information, it can be identified whether there are microorganisms in the sample liquid to be tested. This enables accurate, sensitive, low-cost and rapid detection of microorganisms in blood or body fluids.

In order to solve the above problems, another task of the present invention is to provide a blood analysis method and a blood analyzer that can perform low-cost routine blood testing based on flow cytometry and using fluorescent labeling technology. Simple, sensitive, fast and accurate detection of parasites in blood.

To this end, the fourth aspect of the present invention provides a blood analysis method, including the following steps:

Using a dye reagent containing a first dye and a hemolytic agent for lysing red blood cells to process the same blood sample to be tested to obtain a sample liquid to be tested, wherein the first dye can stain parasites in the blood, and the The first dye includes a compound having the structure of the above general formula I, wherein R ₁ and R ₂ are the same or different, and are independently selected from C _1-18 straight chain or branched alkyl, C _1-18 straight chain or Branched alkylene-M, M is selected from sulfonate group, phenyl, carboxyl, mercapto, amino group, R ₃ is selected from hydrogen, sulfonate group, halogen, cyano group, C _1-6 alkyl group, hydroxyl group, C _{1 -6} alkoxy group, halogenated C _1-6 alkyl group, Y does not exist or is a counter anion;

Parasites in the sample fluid to be tested are identified based on the scattered light information and the first fluorescence information.

In some embodiments, identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes:

Parasites in the sample fluid to be tested are identified based on the first scatter plot.

A parasite characteristic area and a leukocyte area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the parasite characteristic area is greater than the intensity of the first fluorescence information of the leukocyte area; and

Parasites in the sample fluid to be tested are identified based on the parasite characteristic area.

The number of parasites in the sample liquid to be tested is obtained based on the scattered light information and the first fluorescence information.

In some embodiments, the blood analysis method further includes:

In some embodiments, the compound according to the first aspect of the present invention can be used as the compound included in the first dye. For more details and advantages of the compound, please refer to the above description and will not be repeated here.

In some embodiments, the dye reagent further includes a second dye different from the first dye, the second dye is capable of staining white blood cells and nucleated red blood cells in the blood, and the fluorescence information further includes information from the second dye the second fluorescence information; and

The blood analysis method further includes: obtaining at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information.

Identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information includes: identifying parasites in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information. parasites; and

In some embodiments, the dye reagent further includes a second dye that is different from the first dye, the second dye is capable of staining white blood cells in the blood, and the fluorescence information further includes a second fluorescence from the second dye. information; and

The blood analysis method further includes: obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

A fifth aspect of the present invention provides a blood analyzer, including:

Sampling device, used to quantitatively draw blood samples to be tested;

A sample preparation device having a reaction tank and a reagent supply part, wherein the reaction tank is used to receive the blood sample to be tested drawn by the sampling device, and to receive a dye containing a first dye provided by the reagent supply part Reagents and hemolytic reagents for lysing red blood cells. The blood sample to be tested drawn by the sampling device is mixed with the dye reagent and hemolytic reagent provided by the reagent supply part in the reaction tank to prepare a sample to be tested. liquid, wherein the first dye includes a compound with the structure of the above general formula I, wherein R ₁ and R ₂ are the same or different, and are independently selected from C _1-18 linear or branched alkyl, C _{1 -18} Linear or branched alkylene-M, M is selected from sulfonate group, phenyl, carboxyl, mercapto, amino group, R ₃ is selected from hydrogen, sulfonate group, halogen, cyano group, C _1-6 alkyl group , hydroxyl group, C _1-6 alkoxy group, halogenated C _1-6 alkyl group, Y does not exist or is a counter anion;

A processor configured to acquire the scattered light information and the fluorescence information from the optical detection device, and identify parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information.

In some embodiments, the processor is further configured to perform the following steps when identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information:

In some embodiments, the processor is further configured to:

The processor is further configured to: identify parasites in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information, and identify parasites in the sample liquid to be tested based on the forward scattered light information and the second fluorescence information. The fluorescence information obtains at least one of white blood cell count, basophil count and nucleated red blood cell count of the sample fluid to be tested.

The processor is further configured to: identify parasites in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information, and identify parasites in the sample liquid to be tested based on the side scattered light information and the second fluorescence information. The fluorescence information is used to obtain the leukocyte classification result of the sample liquid to be tested.

In the sample analysis method proposed in the fourth aspect of the present invention and the sample analyzer proposed in the fifth aspect, first, a cyanine dye capable of staining parasites, especially a benzothiazole-based cyanine dye and a cyanine dye used to dissolve red blood cells are used. The blood sample to be tested is treated with a hemolytic agent to obtain the sample liquid to be tested, and then flow cytometry is used to obtain the scattered light information and fluorescence information of the sample liquid to be tested. Based on the scattered light information and the fluorescence information, the sample to be tested can be identified. Test the sample fluid for the presence of parasites. This enables accurate, sensitive, low-cost and rapid detection of parasites in blood.

The embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of preferred embodiments.

Description of drawings

Figure 1 is a schematic flow chart of an embodiment of a sample analysis method for identifying microorganisms according to the present invention.

Figure 2 is a first scatter plot when the biological sample to be tested is a blood sample according to an embodiment of the present invention.

Figure 3 is a first scatter plot when the biological sample to be tested is a body fluid sample according to an embodiment of the present invention.

Figure 4 is a schematic flow chart of another embodiment of a sample analysis method for identifying microorganisms according to the present invention.

Figure 5 is a scatter plot of microbial identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to the present invention.

Figure 6 is a schematic flow chart of yet another embodiment of a sample analysis method for identifying microorganisms according to the present invention.

Figure 7 is a scatter plot of microbial identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to the present invention.

Figure 8 is a schematic diagram of the emission spectra of two dyes according to an embodiment of the present invention.

Figure 9 is a schematic diagram of the emission spectrum and excitation spectrum of a large Stokes shift dye according to an embodiment of the present invention.

Figure 10 is a schematic flow chart of an embodiment of a blood analysis method for identifying parasites according to the present invention;

Figure 11 is a first scatter plot for identifying parasites according to an embodiment of the present invention;

Figure 12 is a schematic flow chart of another embodiment of a blood analysis method for identifying parasites according to the present invention;

Figure 13 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to the present invention;

Figure 14 is a schematic flow chart of yet another embodiment of a blood analysis method for identifying parasites according to the present invention;

Figure 15 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to the present invention;

Figure 16 is a schematic structural diagram of an embodiment of a sample analyzer according to the present invention.

17 to 19 are structural schematic diagrams of different embodiments of the optical detection device according to the present invention.

Figure 20 shows the changes in fluorescence spectrum of the dye in Example 7 as the DNA concentration increases.

Figure 21 shows the changes in fluorescence spectrum of the dye in Example 7 as the RNA concentration increases.

Figure 22 is a linear relationship diagram between fluorescence intensity and calf thymus DNA and RNA concentration in Example 7.

Figure 23 shows the results of observing the staining of HepG2 living cells by Compound II under a laser microscope in Example 8.

Figure 24 shows the results of observing the staining of HepG2 living cells by Compound III under a laser microscope in Example 9.

Figure 25 shows the evaluation results of dye stability in Example 10.

Figure 26 shows the evaluation results of the cell penetration of the dye in Example 11.

Figure 27 is a scatter plot of microbial identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 12 of the present invention.

Figure 28 is a scatter plot obtained by testing body fluid samples according to Embodiment 13 of the present invention.

Figure 29 is a scatter plot of microbial identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 14 of the present invention.

Figure 30 is a scatter plot obtained by testing body fluid samples according to Embodiment 15 of the present invention.

Figure 31 is a scatter plot of microbial identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 16 of the present invention.

Figure 32 is a scatter plot obtained by testing body fluid samples according to Embodiment 17 of the present invention.

Figure 33 is a scatter plot of microbial identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to Embodiment 18 of the present invention.

Figure 34 is a scatter plot obtained by testing body fluid samples according to Embodiment 19 of the present invention.

Figure 35 is a scatter plot of microbial identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to Embodiment 20 of the present invention.

Figure 36 is a scatter plot obtained by testing body fluid samples according to Embodiment 21 of the present invention.

Figure 37 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to Embodiment 22 of the present invention.

Figure 38 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to Embodiment 23 of the present invention.

Figure 39 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample according to Embodiment 24 of the present invention.

Figure 40 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 25 of the present invention.

Figure 41 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 26 of the present invention.

Figure 42 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample according to Embodiment 27 of the present invention.

Detailed ways

The embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

It should be noted that the terms "first\second\third" involved in the embodiment of the present invention are only used to distinguish similar objects and do not represent a specific ordering of objects. It is understandable that "first\second\third" Three" may interchange specific order or precedence where permitted.

As an essential instrument for routine blood analysis in modern clinical hematology departments, blood cell analyzers (also known as blood analyzers and hemocytometers) based on flow cytometry have the advantages of rapid, accurate and extremely simple operation for detecting blood cells. It is an indispensable fully automated rapid blood cell detection instrument in modern clinical testing.

Based on this, the present invention proposes a technical solution for rapid detection of pathogenic microorganisms in blood or body fluids based on flow cytometry and fluorescent labeling technology.

In the embodiment of the present invention, microorganisms refer to tiny organisms that are difficult to see clearly with the naked eye and require an optical microscope or an electron microscope to be observed. Microorganisms include bacteria, viruses, fungi and a few algae.

As shown in Figure 1, an embodiment of the present invention first provides a sample analysis method 100 for rapid detection of microorganisms in blood or body fluids based on flow cytometry and fluorescent labeling technology. The sample analysis method 100 includes the following steps S110, S120 and S130.

In step S110, the same biological sample to be tested is processed using a dye reagent containing the first dye and a hemolytic agent for lysing red blood cells to obtain a sample liquid to be tested. Among them, the biological sample to be tested is a blood sample to be tested or a body fluid sample to be tested. Here, the first dye can stain the microorganisms. That is to say, when there are microorganisms in the blood sample to be tested or the body fluid sample to be tested, using the first dye to process the blood sample to be tested or the body fluid sample to be tested can cause the microorganisms therein to be stained.

For example, in step S110, the hemolyzing agent and the dye reagent can be added to the same blood sample or body fluid sample to be tested successively to obtain the sample liquid to be tested, and then the sample liquid to be tested is incubated so that the dye reagent can be fully To stain the substance to be tested in the sample liquid to be tested. For another example, in step S110, the dye reagent may be mixed with the hemolyzing agent in advance to obtain a mixed reagent, and then the mixed reagent and the biological sample to be tested may be mixed at a volume ratio of 250:1-1000:1. After mixing evenly, the mixture The obtained sample liquid to be tested is incubated at a temperature of 25°C to 50°C for 10 seconds to 1 minute, preferably 20 seconds to 40 seconds.

It can be understood that in embodiments of the present invention, the hemolytic agent is used to dissolve red blood cells in blood or body fluids and split the red blood cells into fragments, but can keep the morphology of white blood cells basically unchanged.

In some embodiments, the hemolytic agent may include any one or a combination of cationic surfactants, nonionic surfactants, anionic surfactants, amphiphilic surfactants, and buffer pairs. The cationic surfactant is, for example, selected from at least one or a combination of dodecyltrimethylammonium chloride, octyltrimethylammonium bromide, and tetradecyltrimethylammonium chloride. The nonionic surfactant is, for example, at least one or a combination of several selected from the group consisting of long-chain fatty alcohol polyoxyethylene, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ether, and fatty amine polyoxyethylene ether. The buffer pair is, for example, selected from at least one or a combination of several types of phosphates, citrates, and Tris-HCl. The anionic surfactant is, for example, selected from the group consisting of dodecylbenzene sulfonic acid, sodium fatty alcoholyl sulfate, sodium ethoxylated fatty acid methyl ester sulfonate, sodium secondary alkyl sulfonate, alcohol ether carboxylate at least one or a combination of several.

In other embodiments, the hemolytic agent may include at least one of alkyl glycosides, triterpene saponins, and steroidal saponins.

In step S120, the particles in the sample liquid to be tested are allowed to pass through the optical detection area one by one and the particles flowing through the optical detection area are irradiated with light to obtain the scattered light information and fluorescence generated by the particles in the sample liquid to be tested after being irradiated with light. information. Here, the fluorescence information at least includes the first fluorescence information from the first dye. That is to say, the first fluorescence information includes the fluorescence signal generated under light excitation after the particles in the sample liquid to be tested are combined with the first dye.

That is to say, in step S120, the scattered light information and fluorescence information of the sample liquid to be tested are obtained based on the principle of flow cytometry. When light, such as a laser beam, irradiates particles flowing through the optical detection area, the characteristics of the particles themselves (such as volume, staining degree, cell content size and content, cell nucleus density, etc.) can block or change the direction of the laser beam, resulting in a corresponding The scattered light at various angles corresponding to the characteristics can be received by the signal detector to obtain light information related to the structure and composition of the particles, that is, the scattered light information and fluorescence information of the present invention. Here, the scattered light information includes, for example, at least one of forward scattered light information and side scattered light information. Among them, forward scattered light reflects the number and volume of particles, side scattered light (SS) reflects the complexity of the internal structure of the cell (such as intracellular particles or nuclei), and fluorescence (Fluorescence, FL) reflects the nucleic acid in the cell. substance content. This light information can be used to identify various types of particles in the sample liquid to be tested.

In the embodiment of the present invention, the scattered light information includes scattered light signal intensity, and the fluorescence information includes fluorescence signal intensity.

In step S130, microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

In some embodiments, step S130, that is, identifying microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information may include: generating a first scatter plot based on the scattered light information and the first fluorescence information; and based on the first Scatter plots identify microorganisms in the sample fluid to be tested.

In the embodiment of the present invention, the scatter plot may be a two-dimensional scatter plot or a three-dimensional scatter plot, on which two-dimensional or three-dimensional feature information of multiple particles is distributed, where the X coordinate axis and Y coordinate of the scatter plot Both the axes and the Z coordinate axis represent a characteristic of each particle. For example, in a scatter plot, the X-axis represents the forward scattered light signal intensity, the Y-axis represents the fluorescence signal intensity, and the Z-axis represents the side-scattered light signal intensity. It should be noted that the scatter plots in this article are not limited to graphical form, and can also be in data form, such as numerical forms of tables or lists with the same or similar resolution as the scatter plots, or in any form that has been used in this field. Know of other suitable ways to present it.

Further, the scattered light information may include forward scattered light information FSC, especially forward scattered light signal intensity. Correspondingly, generating the first scattergram based on the scattered light information and the first fluorescence information may include: based on the forward scattered light information FSC, especially the forward scattered light signal intensity and the first fluorescence information FL1, especially the first fluorescence signal. A first scatter plot of intensity is generated, as shown in Figures 2 and 3. Wherein, FIG. 2 shows the first scatter plot when the biological sample to be tested is a blood sample, and FIG. 3 shows the first scatter plot when the biological sample to be tested is a body fluid sample.

Further, as shown in Figures 2 and 3, identifying microorganisms in the sample liquid to be tested based on scattered light information and first fluorescence information may include:

The microbial characteristic area P1 and the white blood cell area P2 are obtained from the first scatter plot, and the intensity of the first fluorescence information of the microbial characteristic area is greater than the intensity of the first fluorescence information of the white blood cell area; and

Microorganisms in the sample liquid to be tested are identified based on the microbial characteristic area P1.

Here, through repeated research by the inventor, in a scatter plot in which the forward scattered light information is the abscissa and the first fluorescence information is the ordinate, such as the two-dimensional scatter plot shown in Figures 2 and 3, in There is a specific area above the white blood cell group (i.e., the white blood cell area) (i.e., the first fluorescence signal intensity of the specific area is not less than the first fluorescence signal of the white blood cell area) Signal intensity), when there are microorganisms in the blood sample or body fluid sample, there are always clusters of scattered points in this characteristic area, and when there are no microorganisms in the blood sample or body fluid sample, there are no clusters of scattered points in this characteristic area. The scattered points of the group. Therefore, clusters of scattered points in this characteristic area can be used to characterize microbial particle clusters, and this characteristic area can also be called a microbial characteristic area. When the number of scatter points in the characteristic area exceeds a predetermined threshold, it is considered that microorganisms are present in the blood sample or body fluid sample to be tested.

In some embodiments, identifying microorganisms in the sample fluid to be tested based on scattered light information and first fluorescence information may include: obtaining microorganisms in the sample fluid to be tested based on scattered light information, especially forward scattered light information and first fluorescence information. Number of microorganisms. For example, in the embodiments shown in Figures 2 and 3, the number of scattered points in the microbial characteristic area P1 can be used to characterize the number of microorganisms in the sample liquid to be tested.

In some feasible specific examples, scatter point data of a large number of biological samples containing microorganisms (such as the number of scatter points in the microbial characteristic area P1) and the actual number of microorganisms in these biological samples can be collected in advance, and the microorganisms can be obtained through fitting The correlation curve between the scatter point data and the actual quantity is obtained to obtain the corresponding calculation model, such as a linear calculation model. In the actual test of the biological sample to be tested, the microbial scatter point data of the biological sample to be tested is obtained according to the method of the present invention. Based on the microbial scatter point data of the biological sample to be tested and the above-mentioned predetermined calculation model, the biological sample to be tested can be estimated. The number of microorganisms in the microorganisms, thereby achieving quantitative analysis of microorganisms.

In some embodiments, when the biological sample to be tested is a blood sample to be tested, the sample analysis method 100 may further include: identifying the sample liquid to be tested based on the scattered light information and the first fluorescence information. The parasites in the sample liquid to be tested, especially malaria parasites, are particularly identified based on the first scatter plot. This enables simultaneous detection of microorganisms and parasites in blood through a single test of the same biological sample to be tested, improving blood detection efficiency and saving reagent costs without increasing blood consumption.

In some embodiments, when the biological sample to be tested is a blood sample to be tested, in step S110, the dye reagent may further include a second dye different from the first dye, and the second dye can affect the blood. cells are stained. At this time, the fluorescence information obtained in step S120 also includes the second fluorescence information FL2 from the second dye, especially the second fluorescence signal intensity. That is, the second fluorescence information includes the particles in the sample liquid to be tested and the second dye. The fluorescence signal generated under light excitation after binding. Thus, the cell parameters of the sample fluid to be tested, such as white blood cell parameters, nucleated red blood cell parameters, etc., can be further obtained based on the scattered light information and the second fluorescence information.

As some implementations, a second dye capable of staining white blood cells in the blood, especially a second dye used for white blood cell classification, is used in step S110. Correspondingly, as shown in FIG. 4 , the sample analysis method 100 may also include step S140: obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information. In a specific example, the white blood cells in the sample fluid to be tested can be classified into a lymphocyte group, a monocyte group, a neutrophil group, and an eosinophil group based on the scattered light information and the second fluorescence information, and the Various types of white blood cells are counted to obtain lymphocyte count and/or the ratio of lymphocyte count to white blood cell count, monocyte count and/or monocyte count to the ratio of white blood cell count, neutrophil count and/or neutrophil count granulocyte count as a ratio of white blood cell count, eosinophil count, and/or eosinophil count as a ratio of white blood cell count. In another example, the white blood cells in the sample fluid to be tested can also be classified into a lymphocyte population, a monocyte population, a neutrophil population, an eosinophil population, and a basophil population based on the scattered light information and the second fluorescence information. granulocyte population, and count various types of white blood cells. This enables simultaneous detection of microorganisms and white blood cells in the blood through one test of the same blood sample to be tested, improving blood detection efficiency and saving reagent costs without increasing blood consumption.

In some embodiments, the second dye used for leukocyte classification can be a nucleic acid dye that can bind to nucleic acid substances in cells, for example, including cyanine cationic compounds. For more details, please refer to the applicant's report on September 28, 2019. Riti Chinese patent application CN101750274A, the entire disclosure content of which is incorporated herein by reference.

In some embodiments, the second dye used for leukocyte classification is a non-DNA or RNA-specific nucleic acid dye, including, for example, a compound of Formula II below.

Further, the scattered light information may include side scattered light information SSC and forward scattered light information FSC. Correspondingly, identifying the microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information, that is, step S130, may include: identifying the microorganisms in the sample liquid to be tested based on the forward scattered light information FSC and the first fluorescence information FL1. , as shown in Figure 5A; and obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information, that is, step S140 may include: obtaining the sample to be tested based on the side scattered light information SSC and the second fluorescence information FL2 The leukocyte classification results of the liquid, such as the four leukocyte classification results as described above, are shown in Figure 5B. For example, a second scatter plot is generated based on the side scattered light information and the second fluorescence information. On the second scatter plot, the white blood cells in the sample fluid to be tested are divided into neutrophils and lymphocytes based on gating technology. population, monocyte population, and eosinophil population and count these cell populations.

Further, step S140 may also include obtaining the leukocyte classification result of the sample liquid to be tested based on the forward scattered light information FSC, the side scattered light information SSC and the second fluorescence information FL2.

As another implementation manner, a second dye capable of staining nucleated cells in the blood, such as leukocytes and nucleated red blood cells, is used in step S110. The second dye can be used in particular to identify nucleated red blood cells. Correspondingly, as shown in Figure 6, the sample analysis method 100 may also include step S150: obtaining the white blood cell count, basophil count and nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information. At least one, especially based on the scattered light information and the second fluorescence information, obtains the white blood cell count, the basophil count and the nucleated red blood cell count of the sample fluid to be tested. This enables simultaneous detection of microorganisms in blood and detection of white blood cells and/or nucleated red blood cells through one test of the same blood sample to be tested, improving blood detection efficiency and saving reagent costs without increasing blood consumption.

In some embodiments, the second dye used for staining nucleated cells can also be a nucleic acid dye that can bind to nucleic acid substances in the cell. However, unlike the above-mentioned dye used for leukocyte classification, it can also be a nucleic acid dye that can bind to intracellular nucleic acid substances. The protein substance binds the protein dye. In one example, the second dye used for staining nucleated cells includes, for example, a compound having the following chemical formula III.

Further, the scattered light information may include forward scattered light information FSC. Correspondingly, identifying microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information, that is, step S130 may include: based on the forward scattered light information FSC and the first fluorescence information FL1 to identify the microorganisms in the sample liquid to be tested, as shown in Figure 7A; and based on the scattered light information and the second fluorescence information, the white blood cell count, basophil count and active granulocyte count of the sample liquid to be tested are obtained. At least one of the nucleated red blood cell count, that is, step S150 may include: obtaining at least one of the white blood cell count, the basophil count, and the nucleated red blood cell count of the sample fluid to be tested based on the forward scattered light information FSC and the second fluorescence information FL2. A, as shown in Figure 7B.

In some embodiments, in the case of using a dye reagent including a first dye and a second dye to process a biological sample to be tested, in step S120, irradiating the particles flowing through the optical detection zone with light includes: using a single wavelength of Light, in particular blue light, for example with a wavelength of around 450 nanometers, illuminates the particles flowing through the optical detection zone.

Here, when one excitation light is used to simultaneously excite two dyes, that is, a first dye and a second dye, in order to better distinguish between collecting the first fluorescence information corresponding to the first dye and the third fluorescence information corresponding to the second dye, Two fluorescence information, thereby more accurately distinguishing leukocytes and microorganisms in blood through two dyes under hemolytic conditions, so that the first dye and the second dye are selected so that the peaks of the emission spectra of the first dye and the second dye correspond The absolute value of the wavelength difference is greater than 30 nanometers and less than 80 nanometers. Alternatively or additionally, the first dye and the second dye are selected such that the emission spectra of the first dye and the second dye overlap by no more than 50%. By selecting such a first dye and a second dye, the detection interference between the first fluorescent signal and the second fluorescent signal can be greatly reduced, that is, the distinction between the first fluorescent signal and the second fluorescent signal can be greatly increased.

Figure 8 shows a schematic diagram of the emission spectra of the first dye and the second dye. The curve represented by the dotted line is the emission spectrum 210 of the first dye, and the curve represented by the solid line is the emission spectrum 220 of the second dye. The peak point of the emission spectrum 210 of the first dye is A, and the peak point of the emission spectrum 220 of the second dye is D. Here, the absolute difference between the abscissas of peak point A and peak point D (ie, the difference in wavelengths corresponding to the peaks) is greater than 30 nanometers and less than 80 nanometers. In addition, the overlap amount of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye may be a ratio of the area of the first polygon to the area of the second polygon, where the area of the first polygon is equal to point E, The area of the curved polygon surrounded by point G and point C, and the area of the second polygon is equal to the curve formed by the emission spectrum 210 of the first dye (or the emission spectrum 220 of the second dye) and the baseline 230 The area of the side polygon, where the reference line 230 is a dotted horizontal line parallel to the horizontal axis as shown in Figure 8. The dotted horizontal line is at the normalized peak of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye. 5%. Points E and F are respectively the left and right intersection points of the emission spectrum 220 of the second dye and the reference line 230. Points B and point C are respectively the left and right intersection points of the emission spectrum 210 of the first dye and the reference line 230. Here, the overlap of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye is no more than 50%.

It is further advantageous that, especially when irradiating with light of a single wavelength, the absolute value of the difference in wavelengths corresponding to the peaks of the emission spectra of the first dye and the second dye is greater than 40 and less than 80 nanometers, preferably greater than 50 nanometers. And less than 80 nanometers, more preferably greater than 50 nanometers and less than 70 nanometers, so as to further reduce the detection interference between the first fluorescence signal and the second fluorescence signal.

In addition, it is advantageous that the overlap of the emission spectra of the first dye and the second dye is no more than 35%, preferably no more than 15%, thereby further reducing the detection interference between the first fluorescence signal and the second fluorescence signal. . Wherein, the smaller the overlap of the emission spectra of the first dye and the second dye, the more conducive it is to distinguish the first fluorescence signal and the second fluorescence signal.

Next, some embodiments of the first dye of the present invention are described.

In some embodiments, the first dye can be a large Stokes shift dye. Here, a large Stokes shift dye refers to a dye whose emission spectrum and excitation spectrum have wavelength differences corresponding to respective peaks greater than a predetermined threshold.

Figure 9 is a schematic spectrum diagram of a large Stokes shift dye. The excitation spectrum (also called absorption spectrum) 310 of the large Stokes shift dye is shown by the dotted line, and the emission spectrum 320 is shown by the solid line. Among them, the excitation spectrum is 310 The peak point of is A1, and the peak point of emission spectrum 320 is A2. The difference between the abscissas of the peak point A2 and the peak point A1 (ie, the difference in wavelengths corresponding to the respective peaks of the emission spectrum and the excitation spectrum) is greater than the predetermined threshold. The predetermined threshold may be, for example, greater than 30 nanometers and less than 150 nanometers, preferably greater than 50 nanometers and less than 100 nanometers.

The use of dyes with a large Stokes shift can in particular reduce mutual detection interference of the first fluorescent signal and the second fluorescent signal.

As some implementations, the first dye is a dye that can specifically bind to deoxyribonucleic acid (ie, DNA), such as a cyanine dye, especially a benzothiazole-based cyanine dye.

In some embodiments, the first dye may include a compound having the structure of Formula I:

Wherein, R ₁ and R ₂ are the same or different, and are independently selected from C _1-18 linear or branched alkyl, C _1-18 linear or branched alkylene-M, and M is selected from sulfonic acid group, Phenyl, carboxyl, mercapto, amino; R ₃ is selected from hydrogen, sulfonic acid group, halogen, cyano, C _1-6 alkyl, hydroxyl, C _1-6 alkoxy, halogenated C _1-6 alkyl; And Y is absent or is a counter anion.

Here, the first dye including a compound with the structure of general formula I proposed by the present invention has one or more of the following advantages: good thermal stability; high biological (microorganism, cell) penetrating power; capable of specificity It combines well with DNA, which is beneficial to the specific recognition and accurate measurement of DNA; it has good permeability to living cells and can enter cells to stain nucleic acids without damaging the cell membrane. It has low toxicity and low carcinogenicity; it can be used It can be excited by blue-green light with a smaller wavelength, so that it can identify tiny particles and improve the detection ability of small particles; it can use ordinary green semiconductor lasers as light sources, which greatly reduces the cost of use; it has a simple structure and the raw materials for its preparation are easy to obtain. The synthesis yield is high and easy to realize industrialization.

In some embodiments, R ₁ and R ₂ are independently selected from C _1-6 linear alkyl, C _1-6 linear alkylene-M, and M is selected from sulfonate, phenyl, carboxyl, mercapto, Amino.

In some embodiments, at least one of R ₁ and R ₂ is a C _1-18 linear or branched alkylene-sulfonic acid group, a C _1-18 linear or branched alkylene-carboxy group.

In some embodiments, R ₁ and R ₂ are different and independently selected from C _1-6 linear alkyl, benzyl, C _1-6 linear alkylene-carboxy, C _1-6 linear alkylene -Sulfonic acid group, C _1-6 linear alkylene-mercapto group, C _1-6 linear alkylene-amino group. In other embodiments, R ₁ and R ₂ are the same and selected from C _1-6 linear alkyl, C _1-6 linear alkylene-sulfonate, C _1-6 linear alkylene-carboxy .

In some embodiments, when R ₃ is hydrogen, R ₁ and R ₂ are not simultaneously methyl and R ₁ and R ₂ are not simultaneously benzyl.

In some embodiments, Y in Formula I is a counter anion. In this case, Y can be selected from halide ions (such as F ^- , Cl ^- , Br ^- , I ^- ), ClO ₄ ^- , PF ₆ ^- , CF ₃ SO ₃ ^- , BF ₄ ^- , acetate, methanesulfonate or p-toluenesulfonate. In other embodiments, Y in Formula I is absent, in which case the compound may be an internal salt. "Internal salts" are also known in the art as "zwitterions". For example, the compounds of the present invention may contain acidic groups (such as sulfonic acid groups or carboxyl group) and a basic group (such as an amino group or a thiazole ring), the acidic group and the basic group neutralize each other to form an internal salt.

In some embodiments, the compound of the present invention having the structure of Formula I may have any structure shown in the following table:

Preferably, the compound of the present invention is a compound represented by the above structural formula 5, 6 or 9.

In other embodiments, in general formula I, R ₁ and R ₂ are each independently selected from C _1-18 alkyl, C _1-18 sulfonic acid group, C _1-18 carboxyl, C _1-18 hydroxyl, The group consisting of C _1-18 NR ₅ R ₆ , benzyl and substituted benzyl, wherein the substituent of the substituted benzyl is selected from C _1-18 alkyl, CN, COOH, NH ₂ , NO ₂ , OH, The group consisting of SH, C _1-6 alkoxy, C _1-6 alkylamino, C _1-6 amido, halogen and C _1-6 haloalkyl, preferably R ₁ and R ₂ are the same C _1-18 sulfonate Acid group; R ₃ is selected from the group consisting of H, C _1-18 sulfonic acid group, phenyl, OR ₆ and halogen; and Y- is a negative ion.

In some embodiments, the dye reagent of the present invention can be stored in a water-soluble organic phase such as glycerol, glycol, and ethylene glycol.

In some embodiments, the dye reagent of the present invention can be stored alone or mixed with a hemolytic agent.

The present invention also proposes a technical solution for rapid detection of parasites in blood based on flow cytometry and fluorescent labeling technology.

In embodiments of the present invention, the parasites are selected from the group consisting of: roundworms, hookworms, tapeworms, Trichomonas vaginalis, liver flukes, Paragonimus westermani, Toxoplasma gondii, cysticercosis suis, Trichinella spiralis, amoeba, Leishmania donovani, Plasmodium, schistosomiasis, filarial worms, hydatid, scabies mites, hair follicle mites, lice, fleas.

As shown in Figure 10, an embodiment of the present invention also provides a blood analysis method 1000 for rapidly detecting parasites in blood based on flow cytometry and fluorescent labeling technology. The blood analysis method 1000 includes the following steps S1100, S1200 and S1300.

In step S1100, the same blood sample to be tested is processed using a dye reagent containing the first dye and a hemolytic agent for lysing red blood cells to obtain a sample liquid to be tested. Here, the first dye can stain parasites in the blood. That is to say, when there are parasites in the blood sample to be tested, using the first dye to process the blood sample to be tested can cause the parasites in the blood sample to be tested to be stained. Here, the first dye is a cyanine dye, especially a cyanine dye of the benzothiazole type and includes compounds having the structure of the general formula I:

Wherein, R ₁ and R ₂ are the same or different, and are independently selected from C _1-18 linear or branched alkyl, C _1-18 linear or branched alkylene-M, and M is selected from sulfonic acid group, Phenyl, carboxyl, mercapto, amino, R ₃ is selected from hydrogen, sulfonic acid group, halogen, cyano, C _1-6 alkyl, hydroxyl, C _1-6 alkoxy, halogenated C _1-6 alkyl, Y is absent or is a counter anion.

For example, in step S1100, a hemolyzing agent and a dye reagent can be added to the same blood sample to be tested successively to obtain a sample liquid to be tested, and then the sample liquid to be tested is incubated so that the dye reagent can fully stain the sample to be tested. The substance to be measured in the liquid. For example, in step S1100, the dye reagent may also be mixed with the hemolytic agent in advance to obtain a mixed mixture. Combine the reagents, then mix the mixed reagents and the blood sample to be tested at a volume ratio of 250:1-1000:1. After mixing evenly, incubate the mixed sample liquid to be tested at a temperature of 25°C-50°C for 10 seconds-1 minutes, preferably 20 seconds to 40 seconds.

The above various embodiments of the hemolytic agent used in step S110 of the method 100 can be correspondingly applied to the hemolytic agent used in step S1100, and will not be described again.

The above various embodiments of the compound having the structure of general formula I used in step S110 of the method 100 can be correspondingly applied to the compound of the first dye used in step S1100, and will not be described again.

In step S1200, the particles in the sample liquid to be tested are allowed to pass through the optical detection area one by one and the particles flowing through the optical detection area are irradiated with light to obtain the scattered light information and fluorescence generated by the particles in the sample liquid to be tested after being irradiated with light. information. Here, the fluorescence information at least includes the first fluorescence information from the first dye. That is to say, the first fluorescence information includes the fluorescence signal generated under light excitation after the particles in the sample liquid to be tested are combined with the first dye.

That is to say, in step S1200, the scattered light information and fluorescence information of the sample liquid to be tested are obtained based on the principle of flow cytometry. When light, such as a laser beam, irradiates particles flowing through the optical detection area, the characteristics of the particles themselves (such as volume, staining degree, cell content size and content, cell nucleus density, etc.) can block or change the direction of the laser beam, resulting in a corresponding The scattered light at various angles corresponding to the characteristics can be received by the signal detector to obtain light information related to the structure and composition of the particles, that is, the scattered light information and fluorescence information of the present invention. Here, the scattered light information includes, for example, at least one of forward scattered light information and side scattered light information. Among them, forward scattered light reflects the number and volume of particles, side scattered light (SS) reflects the complexity of the internal structure of the cell (such as intracellular particles or nuclei), and fluorescence (Fluorescence, FL) reflects the nucleic acid in the cell. substance content. This light information can be used to identify various types of particles in the sample liquid to be tested.

In step S1300, parasites in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

In some embodiments, step S1300, that is, identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information, may include: generating a first scatter plot based on the scattered light information and the first fluorescence information; and based on the first scattered light information and the first fluorescence information. A scatter plot identifies parasites in the sample fluid being tested.

Further, the scattered light information may include forward scattered light information FSC, especially forward scattered light signal intensity. Correspondingly, generating the first scattergram based on the scattered light information and the first fluorescence information may include: based on the forward scattered light information FSC, especially the forward scattered light signal intensity and the first fluorescence information FL1, especially the first fluorescence signal. A first scatter plot of intensity is generated, as shown in Figure 11.

Further, as shown in Figure 11, identifying parasites in the sample liquid to be tested based on scattered light information and first fluorescence information may include:

The parasite characteristic area P1 and the white blood cell area P2 are obtained from the first scatter plot, and the intensity of the first fluorescence information of the parasite characteristic area is greater than the intensity of the first fluorescence information of the white blood cell area; and

Parasites in the sample liquid to be tested are identified based on the parasite characteristic area P1.

Here, through repeated research by the inventor, in a scatter plot in which the forward scattered light information is the abscissa and the first fluorescence information is the ordinate, for example, the two-dimensional scatter plot shown in Figure 11, in the white blood cell group ( That is, there is a specific area above the white blood cell area (that is, the first fluorescence signal intensity of this specific area is not less than the first fluorescence signal intensity of the white blood cell area). When there are parasites in the blood sample, there is always a certain area in this characteristic area. There are clumps of scattered spots, whereas there are no clumps of scattered spots in the characteristic area when parasites are not present in the blood sample. Therefore, clusters of scattered points in this characteristic area can be used to characterize parasite particle clusters, and this characteristic area can also be called a parasite characteristic area. When the feature area When the number of scatter points exceeds a predetermined threshold, the parasite is considered present in the blood sample to be tested.

In some embodiments, identifying parasites in the sample liquid to be tested based on scattered light information and first fluorescence information may include: obtaining parasites in the sample liquid to be tested based on scattered light information, especially forward scattered light information and first fluorescence information. number of parasites. For example, in the embodiment shown in FIG. 11 , the number of scattered points in the parasite characteristic area P1 can be used to characterize the number of parasites in the sample liquid to be tested.

In some feasible specific examples, scatter point data of a large number of parasite-containing blood samples (such as the number of scatter points in the parasite characteristic area P1) and the actual number of parasites in these blood samples can be collected in advance, and the data can be simulated by The correlation curve between the scatter point data of the parasite and the actual number is obtained by combining it, thereby obtaining the corresponding calculation model, such as a linear calculation model. In the actual test of the blood sample to be tested, the parasite scatter point data of the blood sample to be tested is obtained according to the method of the present invention. Based on the parasite scatter point data of the blood sample to be tested and the above-mentioned predetermined calculation model, the parasite scatter point data of the blood sample to be tested can be estimated. The number of parasites in the blood sample, enabling quantitative analysis of parasites.

In some embodiments, the blood analysis method 1000 may further include: identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information, especially based on the first scatter plot. Identify microorganisms in the sample fluid to be tested. This enables simultaneous detection of microorganisms and parasites in the blood through one test of the same blood sample to be tested, improving blood detection efficiency and saving reagent costs without increasing blood consumption.

In some embodiments, in step S1100, the dye reagent may further include a second dye different from the first dye, and the second dye can stain cells in the blood. At this time, the fluorescence information obtained in step S1200 also includes the second fluorescence information FL2 from the second dye, especially the second fluorescence signal intensity. That is, the second fluorescence information includes the particles in the sample liquid to be tested and the second dye. The fluorescence signal generated under light excitation after binding. Thus, the cell parameters of the sample fluid to be tested, such as white blood cell parameters, nucleated red blood cell parameters, etc., can be further obtained based on the scattered light information and the second fluorescence information.

As some implementations, a second dye that can stain nucleated cells in the blood, such as leukocytes and nucleated red blood cells, is used in step S1100. The second dye can be used in particular to identify nucleated red blood cells. Correspondingly, as shown in Figure 12, the blood analysis method 1000 may also include step S1400: obtaining the white blood cell count, basophil count and nucleated red blood cell count of the sample liquid to be tested based on the scattered light information and the second fluorescence information. At least one, especially based on the scattered light information and the second fluorescence information, obtains the white blood cell count, the basophil count and the nucleated red blood cell count of the sample fluid to be tested. As a result, it is possible to simultaneously detect parasites in blood and detect leukocytes and/or nucleated red blood cells through one test of the same blood sample to be tested, thereby improving blood detection efficiency and saving reagent costs without increasing blood consumption.

In some embodiments, the second dye used to stain nucleated cells may be a nucleic acid dye that can bind to nucleic acid substances in the cell, or a protein dye that can bind to protein substances in the cell. In one example, the second dye used for staining nucleated cells includes, for example, a compound having the following chemical formula III.

Further, the scattered light information may include forward scattered light information FSC. Correspondingly, identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information, that is, step S1300 may include: identifying parasites in the sample liquid to be tested based on the forward scattered light information FSC and the first fluorescence information FL1. Parasites, as shown in Figure 13A; and obtaining at least one of the white blood cell count, basophil count, and nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information, that is, step S1400 may include: At least one of the white blood cell count, the basophil count, and the nucleated red blood cell count of the sample fluid to be tested is obtained based on the forward scattered light information FSC and the second fluorescence information FL2, as shown in FIG. 13B .

As another implementation manner, a second dye capable of staining white blood cells in the blood, especially a second dye used for white blood cell classification, is used in step S1100. Correspondingly, as shown in Figure 14, the blood analysis method 1000 may also include step S1500: obtaining the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information. In a specific example, the white blood cells in the sample fluid to be tested can be classified into a lymphocyte group, a monocyte group, a neutrophil group, and an eosinophil group based on the scattered light information and the second fluorescence information, and the Various types of white blood cells are counted to obtain lymphocyte count and/or the ratio of lymphocyte count to white blood cell count, monocyte count and/or monocyte count to the ratio of white blood cell count, neutrophil count and/or neutrophil count granulocyte count as a ratio of white blood cell count, eosinophil count, and/or eosinophil count as a ratio of white blood cell count. In another example, the white blood cells in the sample fluid to be tested can also be classified into a lymphocyte population, a monocyte population, a neutrophil population, an eosinophil population, and a basophil population based on the scattered light information and the second fluorescence information. granulocyte population, and count various types of white blood cells. As a result, it is possible to simultaneously detect parasites and leukocytes in the blood through one test of the same blood sample to be tested, improving blood detection efficiency and saving reagent costs without increasing blood consumption.

In some embodiments, the second dye used for leukocyte classification can also be a nucleic acid dye that can bind to nucleic acid substances in cells, for example, including cyanine cationic compounds. For more details, please refer to the applicant's report in September 2019. The entire disclosure content of the Chinese patent application CN101750274A submitted on the 28th is incorporated herein by reference.

Further, the scattered light information may include side scattered light information SSC and forward scattered light information FSC. Correspondingly, identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information, that is, step S1300, may include: Identify parasites in the sample liquid to be tested based on the forward scattered light information FSC and the first fluorescence information FL1, as shown in Figure 15A; and obtain the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information, that is, Step S1500 may include: obtaining the white blood cell classification result of the sample liquid to be tested based on the side scattered light information SSC and the second fluorescence information FL2, such as the four white blood cell classification results as described above, as shown in Figure 15B. For example, a second scatter plot is generated based on the side scattered light information and the second fluorescence information. On the second scatter plot, the white blood cells in the sample fluid to be tested are divided into neutrophils and lymphocytes based on gating technology. population, monocyte population, and eosinophil population and count these cell populations.

Further, step S1500 may also include obtaining the leukocyte classification result of the sample liquid to be tested based on the forward scattered light information FSC, the side scattered light information SSC and the second fluorescence information FL2.

In some embodiments, in the case where the blood sample to be tested is processed using a dye reagent including a first dye and a second dye, in step S1200, irradiating the particles flowing through the optical detection zone with light includes: using a single wavelength of Light, in particular blue light, for example with a wavelength of around 4500 nanometers, illuminates the particles flowing through the optical detection zone.

Here, when one excitation light is used to simultaneously excite two dyes, that is, a first dye and a second dye, in order to better distinguish between collecting the first fluorescence information corresponding to the first dye and the third fluorescence information corresponding to the second dye, Two fluorescence information, thereby more accurately distinguishing leukocytes and parasites in blood through two dyes under hemolysis conditions, so that the first dye and the second dye are selected so that the peaks of the emission spectra of the first dye and the second dye are The absolute value of the corresponding wavelength difference is greater than 30 nanometers and less than 80 nanometers. Alternatively or additionally, the first dye and the second dye are selected such that the emission spectra of the first dye and the second dye overlap by no more than 50%, see Figure 8 . By selecting such a first dye and a second dye, the detection interference between the first fluorescent signal and the second fluorescent signal can be greatly reduced, that is, the distinction between the first fluorescent signal and the second fluorescent signal can be greatly increased.

In some embodiments, the first dye can be a large Stokes shift dye. Here, a large Stokes shift dye refers to a dye whose emission spectrum and excitation spectrum have wavelength differences corresponding to respective peaks greater than a predetermined threshold. Referring again to FIG. 9 , the difference between the abscissas of the peak point A2 and the peak point A1 is greater than the predetermined threshold. The predetermined threshold may be, for example, greater than 30 nanometers and less than 150 nanometers, preferably greater than 50 nanometers and less than 100 nanometers. The use of dyes with a large Stokes shift can in particular reduce mutual detection interference of the first fluorescent signal and the second fluorescent signal.

For other embodiments of the blood analysis method 1000 provided by the present invention, reference may be made to the above description of the sample analysis method 100 .

The present invention also provides a sample analyzer 400 based on flow cytometry and fluorescent labeling technology. As shown in FIG. 16 , the sample analyzer 400 includes a sampling device 410 , a sample preparation device 420 , an optical detection device 430 and a processor 440 . The sample analyzer 400 also includes a liquid circuit system (not shown) for communicating with the sampling device 410, the sample preparation device 420, and the optical detection device 430 to facilitate liquid transmission between these devices.

The sampling device 410 is used to quantitatively absorb biological samples to be tested, where the biological samples to be tested are blood samples to be tested or body fluid samples to be tested. For example, the sampling device 410 has a pipette with a pipette nozzle and a driving device, which is used to drive the pipette to quantitatively suck the biological sample to be tested through the pipette nozzle. The sampling device can collect the collected organisms to be tested The sample is transported to sample preparation device 420.

The sample preparation device 420 has a reaction cell and a reagent supply part. The reaction pool is used to receive the biological sample to be tested sucked by the sampling device 410, and to receive the dye reagent containing the first dye and the hemolytic agent for dissolving red blood cells provided by the reagent supply unit. The biological sample to be tested sucked by the sampling device 410 and The dye reagent and hemolytic reagent provided by the reagent supply department are mixed in the reaction tank to prepare the sample liquid to be tested.

According to the first embodiment of the present invention, the first dye can dye microorganisms. In this first embodiment, for more embodiments of the first dye, reference can be made to the various embodiments of the first dye used in the sample analysis method 100 above, which will not be described again here.

According to a second embodiment of the invention, the first dye is capable of staining parasites and includes a compound having the structure of general formula I:

In this second embodiment, further examples of the first dye may refer to the above various embodiments of the first dye used in the sample analysis method 100 and the various embodiments of the first dye used in the blood analysis method 1000. The embodiments will not be described again here.

The optical detection device 430 includes a light source, a flow chamber, a scattered light detector and a fluorescence detector. The light source is used to emit a light beam to illuminate the flow chamber. The flow chamber is connected to the reaction cell and the particles in the sample liquid to be measured can pass through the flow chamber one by one, scattering The light detector is used to detect the scattered light information generated by the particles passing through the flow chamber after being illuminated by light, and the fluorescence detector is used to detect the fluorescence information generated by the particles passing through the flow chamber after being illuminated by light. The fluorescence information includes the first dye from the first dye. a fluorescence information.

As used herein, a flow cell refers to a chamber of a focused fluid flow suitable for detecting light scattering signals and fluorescence signals. When a particle, such as a blood cell, passes through the detection hole of the flow chamber, the particle scatters the incident light beam from the light source directed to the detection hole in all directions. Photodetectors may be positioned at one or more different angles relative to the incident light beam to detect light scattered by the particles to obtain a light scattering signal. Since different particles have different light scattering properties, the light scattering signal can be used to distinguish different particle populations. Specifically, the light scattering signal detected near the incident light beam is often referred to as the forward light scattering signal or the small angle light scattering signal. In some embodiments, the forward light scatter signal can be detected from an angle of about 1° to about 10° from the incident light beam. In other embodiments, the forward light scatter signal may be detected from an angle of about 2° to about 6° from the incident light beam. Light scattering signals detected at approximately 90° to the incident beam are often referred to as side light scattering signals. In some embodiments, the side light scatter signal may be detected from an angle of about 65° to about 115° with respect to the incident light beam. Typically, fluorescent signals from blood cells stained with fluorescent dyes are also detected at approximately 90° to the incident light beam.

In some embodiments, the optical detection device 430 includes a forward scattered light detector for detecting forward scattered light or a side scattered light detector for detecting side scattered light. Optical detection device 430 preferably includes forward scattered light detection detector and side scattered light detector.

FIG. 17 shows a specific example of the optical detection device 430. The optical detection device 430 has a light source 401, a beam shaping component 402, a flow chamber 403 and a forward scattered light detector 404 arranged in sequence on a straight line. On one side of the flow chamber 403, a dichroic mirror 406 is arranged at an angle of 45° to the straight line. Part of the lateral light emitted by the particles in the flow chamber 403 passes through the dichroic mirror 406 and is captured by the fluorescence detector 405 arranged behind the dichroic mirror 106 at an angle of 45° to the dichroic mirror 406; the other part The side light is reflected by the dichroic mirror 406 and captured by a side scattered light detector 407 arranged in front of the dichroic mirror 406 at an angle of 45°.

The processor 440 is used to process the optical signals collected by the optical detection device 430 to obtain the required results. For example, a two-dimensional scatter plot or a three-dimensional scatter plot can be generated based on various collected optical signals, and the scatter plot can be generated in the scatter plot. In the figure, particle analysis is performed based on the gating method. The processor 440 can also perform visual processing on the intermediate operation result or the final operation result, and then display it through the display device 450 .

In some embodiments, the processor 440 includes, but is not limited to, a central processing unit (Central Processing Unit, CPU), a micro control unit (Micro Controller Unit, MCU), a field-programmable gate array (Field-Programmable Gate Array, FPGA), a digital A device such as a signal processor (DSP) used to interpret computer instructions and process data in computer software. For example, the processor 440 is used to execute each computer application program in a computer-readable storage medium, so that the sample analyzer 400 executes a corresponding detection process and analyzes the optical signal detected by the optical detection device 430 in real time.

In addition, the sample analyzer 400 also includes a first housing 460 and a second housing 470 . The display device 450 may be, for example, a user interface. The optical detection device 130 and the processor 140 are disposed inside the second housing 170 . The sample preparation device 420 is, for example, disposed inside the first casing 460 , and the display device 450 is, for example, disposed on the outer surface of the first casing 460 and is used to display the detection results of the blood cell analyzer. In other embodiments, a computer having a display may be remotely communicatively connected to the sample analyzer 400, such as being installed remotely from the laboratory where the blood cell analyzer is located, such as in a doctor's office.

According to the first embodiment of the present invention, the processor 440 is configured to obtain scattered light information and fluorescence information from the optical detection device 430, and identify microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information.

In some variations of the first embodiment, the processor 440 may be further configured to perform the following steps when identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information:

Microorganisms in the sample liquid to be tested are identified based on the first scatter plot.

In some variations of the first embodiment, the scattered light information includes forward scattered light information, in which case the processor 440 may be further configured to generate a first scatter plot based on the forward scattered light information and the first fluorescence information.

In some variations of the first embodiment, the processor 440 may also be configured to:

The microbial characteristic area and the white blood cell area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the microbial characteristic area is greater than the intensity of the first fluorescence information of the white blood cell area; and

Identify microorganisms in the sample liquid to be tested based on the microbial characteristic area.

In some variations of the first embodiment, the processor 440 may be further configured to perform the following steps when identifying microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information: based on the scattered light information and the first fluorescence information. The information obtains the number of microorganisms in the sample liquid to be tested.

In some variations of the first embodiment, the processor 440 may be further configured to: identify parasites, especially Plasmodium, in the sample fluid to be tested based on the scattered light information and the first fluorescence information.

In some variations of the first embodiment, the biological sample to be tested is a blood sample to be tested, and the dye reagent may further include a second dye different from the first dye, and the second dye can stain cells in the blood. . At this time, the fluorescence information obtained by the optical detection device 430 also includes second fluorescence information from the second dye. Thus, the cell parameters of the sample fluid to be tested, such as white blood cell parameters, nucleated red blood cell parameters, etc., can be further obtained based on the scattered light information and the second fluorescence information.

FIG. 18 shows another specific example of the optical detection device 430. The optical detection device 430 has a laser 431, a front light assembly 432, a flow chamber 433, a forward scattered light detector 434, a first dichroic mirror 435, a side scattered light detector 436, a second dichroic mirror 437, A first fluorescence detector 438 and a second fluorescence detector 439. The first fluorescence detector 438 is used to detect the first fluorescence signal corresponding to the first dye generated by the particles passing through the flow chamber 433 after being irradiated with light, and the second fluorescence detector 439 is used to detect when the particles passing through the flow chamber 433 are irradiated by light. A second fluorescent signal corresponding to the second dye is generated after light irradiation. Here, the laser 431, the front light assembly 432, the flow chamber 433 and the forward scattered light detector 434 are sequentially arranged on the optical axis along the optical axis direction, and the front light assembly is configured to cause the excitation light emitted by the laser 431 to flow in the particles. The particles converge in the detection area of the flow chamber 433 in the direction, so that the particles flowing through the detection area of the flow chamber 433 can generate scattered light. On one side of the flow chamber 433, a first dichroic mirror 435 is arranged at an angle of 45° to the optical axis. A part of the side light generated by the particles when flowing through the detection area of the flow chamber 433 is reflected by the first dichroic mirror 435 and captured by the side scattered light detector 436, while the other part of the side light passes through the first dichroic mirror 435. The dichroic mirror 435 reaches a second dichroic mirror 437 , which is also arranged downstream of the first dichroic mirror 435 at an angle of 45° to the optical axis. A part of the lateral light that passes through the first dichroic mirror 435 is reflected by the second dichroic mirror 437 and captured by the first fluorescence detector 438, while the other part passes through the second dichroic mirror 437 and is captured by the second fluorescence detector. Detector 439 captures.

In other embodiments, as shown in FIG. 19 , unlike the optical detection device shown in FIG. 18 , the forward scattered light detector 434 may also be arranged tilted to the optical axis. On the optical axis, a reflective mirror 4341 is arranged downstream of the flow chamber along the optical axis direction, which reflects the forward scattered light of the particles into a forward scattered light detector 434 arranged obliquely to the optical axis.

In some variations of the first embodiment, the second dye is a dye capable of staining leukocytes in the blood, in particular a dye used for leukocyte classification. Correspondingly, the processor 440 may also be configured to: obtain the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

Further, the scattered light information includes side scattered light information and forward scattered light information. Correspondingly, the processor 440 may also be configured to: identify microorganisms in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information, and obtain the microorganisms in the sample liquid to be tested based on the side scattered light information and the second fluorescence information. WBC differential results.

In other variations of the first embodiment, the second dye is a dye capable of staining leukocytes and nucleated red blood cells in the blood, in particular a dye for identifying nucleated red blood cells. Correspondingly, the processor 440 may be further configured to: obtain at least one of the white blood cell count, the basophil count, and the nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information.

Further, the scattered light information includes forward scattered light information. Correspondingly, the processor 440 may also be configured to: identify microorganisms in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information, and obtain the microorganisms in the sample liquid to be tested based on the forward scattered light information and the second fluorescence information. At least one of a white blood cell count, a basophil count, and a nucleated red blood cell count.

Preferably, the light source may be configured to illuminate the flow chamber with a single wavelength of light. For example, the optical detection device 430 only has one laser source that emits blue-green light, especially a laser source that emits blue light.

For more embodiments and advantages of the sample analyzer 400 according to the first embodiment of the present invention, please refer to the above description of the sample analysis method 100 of the present invention, and will not be described again here.

According to the second embodiment of the present invention, the processor 440 is configured to obtain scattered light information and fluorescence information from the optical detection device 430, and identify parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information.

In some variations of the second embodiment, the processor 440 may be further configured to perform the following steps when identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information:

Parasites in the sample liquid to be tested are identified based on the first scatter plot.

In some variations of the second embodiment, the scattered light information includes forward scattered light information, in which case the processor 440 may be further configured to generate a first scatter plot based on the forward scattered light information and the first fluorescence information.

In some variations of the second embodiment, the processor 440 may also be configured to:

The parasite characteristic area and the white blood cell area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the parasite characteristic area is greater than the intensity of the first fluorescence information of the white blood cell area; and

Identify parasites in the sample fluid to be tested based on parasite characteristic areas.

In some variations of the second embodiment, the processor 440 may be further configured to perform the following steps when identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information: based on the scattered light information and the first fluorescence information. Fluorescence information is used to obtain the number of parasites in the sample liquid to be tested.

In some variations of the second embodiment, the processor 440 may be further configured to: identify microorganisms in the sample fluid to be tested based on the scattered light information and the first fluorescence information.

In some variations of the second embodiment, the dye agent may further comprise a second dye different from the first dye, the second dye being capable of staining cells in the blood. At this time, the fluorescence information obtained by the optical detection device 430 also includes second fluorescence information from the second dye. Thus, the cell parameters of the sample fluid to be tested, such as white blood cell parameters, nucleated red blood cell parameters, etc., can be further obtained based on the scattered light information and the second fluorescence information.

In some variations of the second embodiment, the second dye is a dye capable of staining white blood cells and nucleated red blood cells in the blood, in particular a dye used to identify nucleated red blood cells. Correspondingly, the processor 440 may be further configured to: obtain at least one of the white blood cell count, the basophil count, and the nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information.

Further, the scattered light information includes forward scattered light information. Correspondingly, the processor 440 may also be configured to: identify parasites in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information, and obtain the sample liquid to be tested based on the forward scattered light information and the second fluorescence information. At least one of a white blood cell count, a basophil count, and a nucleated red blood cell count.

In other variations of the second embodiment, the second dye is a dye capable of staining leukocytes in the blood, in particular a dye used for leukocyte classification. Correspondingly, the processor 440 may also be configured to: obtain the leukocyte classification result of the sample liquid to be tested based on the scattered light information and the second fluorescence information.

Further, the scattered light information includes side scattered light information and forward scattered light information. Correspondingly, the processor 440 may also be configured to: identify parasites in the sample liquid to be tested based on the forward scattered light information and the first fluorescence information, and obtain the sample liquid to be tested based on the side scattered light information and the second fluorescence information. WBC classification results.

For more embodiments and advantages of the blood analyzer 400 according to the second embodiment of the present invention, please refer to the above description of the sample analysis method 100 and the blood analysis method 1000 of the present invention, and will not be described again here.

The present invention also proposes that DNA-specific dyes (that is, dyes that can specifically bind to deoxyribonucleic acid) are used in flow cytometry. Cytometry is used to identify microorganisms in blood samples to be tested.

In some embodiments, DNA-specific dyes include compounds having the structure of Formula I:

For more examples and advantages of the DNA-specific dye proposed by the present invention, please refer to the above description of the sample analysis method 100 of the present invention, and will not be described again here.

The present invention also proposes the application of DNA-specific dyes (ie dyes that can specifically bind to deoxyribonucleic acid) in identifying parasites in blood samples to be tested using flow cytometry. Wherein, the DNA-specific dyes include compounds with the structure of general formula I:

For more examples and advantages of the DNA-specific dye proposed by the present invention, please refer to the above description of the sample analysis method 100 and blood analysis method 1000 of the present invention, and will not be described again here.

The invention will now be described with reference to the following examples which are intended to illustrate but not to limit the invention. Unless otherwise indicated, the experiments and methods described in the examples were performed essentially according to conventional methods well known in the art and described in various references. In addition, if the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1 Synthesis of Compound I

The structure of compound I can be represented by structural formula 1.

In the first step, compound (E)-4-2-(5-formyl-2-hydroxystyryl)benzothiazole-3-butyl-1-sulfonic acid inner salt (structure See the right side of reaction equation I).

Add 5mL methanol to the reaction bottle, and then add 1.33mmol 4-hydroxyisophthalaldehyde (Shanghai Bide Pharmaceutical Technology Co., Ltd.), 0.67mmol (E)-4-(2-(5-formyl-2- Hydroxystyryl)benzothiazol-3-yl)-butyl-1-sulfonate and 2.66 mmol pyridine (Shanghai Bide Pharmaceutical Technology Co., Ltd.). The reaction was stirred at 80°C for 43 hours.

The reaction mixture was filtered with suction, and the filter cake was washed three times with 10 mL of ethanol. The filter cake was collected and dried under reduced pressure to obtain 0.44 mmol of yellow solid powder, which was (E)-4-(2-(5-methyl). Acyl-2-hydroxystyryl)benzothiazol-3-yl)-butyl-1-sulfonic acid inner salt. The yield of reaction scheme I is approximately 33%.

NMR testing was performed on the compound and the results are as follows.

1H NMR (400MHz, DMSO-d6)δ＝12.04(s,1H),9.95(s,1H),8.78(d,J＝2.0Hz,1H),8.42-8.40(m,2H),8.34-8.24( m,2H),7.94-7.86(m,2H),7.80(t,J＝7.6Hz,1H),7.18-7.12(m,1H),5.00-4.88(m,2H),2.59–2.56(m, 2H),2.08-2.10(m,2H),1.89-1.79(m,2H).

The second step is to prepare 4-(2-((E)-2-((E)-3-((Z)))-2-(3-methylbenzothiazole-2(3H)) according to the following reaction formula II -Alkenyl)vinyl)-6-oxo-1,4-cyclohexadien-1-yl)ethyl)benzothiazol-3-yl)butanesulfonate inner salt (Compound I).

Add 5 mL acetic acid to the reaction bottle, and add 0.44 mmol of (E)-4-(2-(5-formyl-2-hydroxystyryl)benzothiazol-3-yl)-butyl- obtained in the second step. 1-Sulfonic acid inner salt (the structure is shown on the left side of reaction formula II), 0.72mmol 2,3 dimethylbenzothiazole salt (Shanghai Haohong Biomedical Technology Co., Ltd.) and 1.58mmol sodium acetate were added to the reaction solution in sequence. In a bottle, react at 110°C for 12 hours.

The reaction mixture was filtered, and the filter cake was washed three times with 5 mL of acetonitrile/water = 1:1 mixed solution to obtain a crude product.

The above crude product was purified by preparative HPLC to obtain about 0.01 mmol red solid powder, which is 4-(2-((E)-2-((E)-3-((Z)))-2- (3-methylbenzothiazole-2(3H)-alkenyl)ethenyl)-6-oxo-1,4-cyclohexadien-1-yl)ethenyl)benzothiazol-3-yl) Butanesulfonate inner salt (Compound I). The yield of reaction scheme II is about 20%.

NMR testing was performed on the product and the results are as follows:

1H NMR (400MHz, DMSO-d6)δ=8.35-8.14(m,4H),8.09-7.94(m,3H),7.91-7.82(m,2H),7.77-7.74(m,1H),7.70-7.63 (m,2H),7.59-7.55(m,1H),7.44-7.29(m,1H),6.39(d,J＝9.2Hz,1H),4.68-4.66(m,2H),4.15-4.13(m ,3H),2.62-2.58(m,2H),2.01-1.92(m,2H),1.85-1.81(m,2H).

After verification, the test results are consistent with the structure of structural formula 1.

Example 2 Synthesis of Compound II

The structure of compound II can be represented by structural formula 2.

In the first step, 2-methyl-3-(butylsulfonic acid) benzothiazole salt is prepared according to the following reaction formula I (the structure is shown on the right side of reaction formula I).

Measure 50 mL of toluene in the container, and add 34 mmol of benzothiazole (left side of reaction formula I) and 40 mmol of 1,4-butanesultone (Shanghai Haohong Biomedical Technology Co., Ltd.) into the container. Reflux and stir for 24 hours under nitrogen protection, and then stop the reaction.

The reaction mixture was suction filtered, and then the filter cake was washed three times with 50 mL of ethyl acetate to obtain a yellow solid product, which was 2-methyl-3-(butylsulfonic acid) benzothiazole salt. The yield of Scheme I is approximately 62%.

In the second step, compound II (4-(2-((E)-6-oxo-3-((Z))-2-(3-(4-sulfobutyl))benzo [d]thiazol-2-ylidene)ethylene)cyclohexan-1,4-dien-1-yl)vinyl)benzo[d]thiazol-3-yl)butanesulfonic acid.

Measure 30 mL of acetic acid into a container, add 1.7 mmol of 2-methyl-3-(butylsulfonate) benzothiazole salt (structure shown above the arrow in Reaction Formula II) and 0.7 mmol of 4 -Hydroxyisophthalaldehyde and 2.2 mmol of sodium acetate were added to the container. The reaction was stirred for 24 hours under nitrogen protection and a temperature of 80°C.

Pour the reacted mixture into 50 mL of petroleum ether:ethyl acetate=5:1 solution prepared in advance, and then pour out the supernatant to obtain the crude product. After the above crude product was beaten with 10 mL of acetonitrile to water equal to 1:1, about 0.34 mmol of brown solid powder was obtained. The brown solid powder was compound II. The yield of reaction formula II was about 52%.

NMR testing was performed on the product and the results are as follows.

1H NMR (400MHz, DMSO-d6, TMS) δ = 8.87 (bs, 1H), 8.42-8.34 (m, 4H), 8.30-8.22 (m, 2H), 8.21-8.07 (m, 3H), 7.88-7.73 (m,4H),7.09(d,J＝8.0Hz,1H),5.00-4.95(m,4H),2.68-2.65(m,4H),2.11-2.03(m,4H),1.90-1.84(m ,4H).

After verification, the test results are consistent with the structure of structural formula 2.

Example 3 Synthesis of Compound VI

The structure of compound VI can be represented by structural formula 6, where Y- is an iodide ion.

In the first step, 2,3-dimethyl-5-chlorobenzothiazole quaternary ammonium iodide is prepared according to the following reaction formula I

20 mL of methyl iodide was added to the reaction flask, followed by 10.9 mmol of 5-chloro-2-methylbenzothiazole (Shanghai Bide Pharmaceutical Technology Co., Ltd.). The reaction was heated to 80°C and stirred at this temperature for 16 hours.

The reaction mixture was filtered, and the filter cake was washed three times with 10 mL of ethyl acetate. The filter cake was collected and dried under reduced pressure to obtain 7.35 mmol of white powder solid. The white powdery solid is 2,3-dimethyl-5-chlorobenzothiazole quaternary ammonium iodide (right side of reaction formula I). The yield of Scheme I is approximately 67%.

NMR testing was performed on the product and the results are as follows.

1H NMR (400MHz, DMSO-d6) δ = 8.53 (d, J = 12.0Hz, 1H), 8.45 (d, J = 8.8Hz, 1H), 7.88 (dd, J = 2.0, 8.8Hz, 1H), 4.18 (s,3H),3.17(s,3H)

In the second step, prepare 5-chloro-2-((E)-2-((E)-3-((Z)-2-(5-chloro-3-methylbenzothiazole)) according to the following reaction formula II -2(3H)-ylidene)vinyl)-6-oxocyclohexan-1,4-dien-1-yl)vinyl-3-methylbenzothiazolium salt (Compound VI)

Measure 6 mL of acetic acid into a container, and add 1.67 mmol of 5-chloro-2,3-dimethylbenzothiazole quaternary ammonium salt (the structure is shown on the left side of Reaction Formula II) obtained in the first step of the reaction to 0.67 mmol of In a mixture of 4-hydroxyisophthalaldehyde (Shanghai Haohong Biomedical Technology Co., Ltd.) and 2.2 mmol sodium acetate. The reaction was stirred at 110°C for 12 hours.

After the reaction is completed, the reaction mixture is filtered, and the filter cake is collected to obtain a crude product.

The above crude product was added to 20 mL of acetonitrile and stirred at 90°C for 2 hours. The solid was collected by filtration to obtain about 0.46 mmol of brown-black solid. The brown-black solid powder was 5-chloro-2-((E)-2-((E)-3-((Z)-2-(5-) Chloro-3-methylbenzothiazole-2(3H)-ylidene)vinyl)-6-oxocyclohexan-1,4-dien-1-yl)vinyl-3-methylbenzothiazole Salt (Compound VI), the yield of reaction formula II is about 70%.

NMR testing was performed on the product and the results are as follows.

1H NMR (400MHz, DMSO-d6) δ = 8.73 (d, J = 1.2Hz, 1H), 8.53 (d, J = 2.0Hz, 1H), 8.47-8.41 (m, 3H), 8.26-8.12 (m, 4H),7.95(d,J＝16.4Hz,1H),7.90-7.84(m,2H),7.16(d,J＝8.8Hz,1H),4.36(d,J＝6.8Hz,6H).

After verification, the test results are consistent with the structure of structural formula 6.

Example 4 Synthesis of Compound VII

The structure of compound VII can be represented by structural formula 7.

In the first step, 3-(5-carboxylic acid pentyl)-2-methylbenzothiazole bromide is prepared according to the following reaction formula I (the structure is shown on the right side of reaction formula I).

67 mmol of benzothiazole (Shanghai Haohong Biomedical Technology Co., Ltd.) (the structure is shown on the left side of Reaction Formula I) was added to the container. Then, 73.72 mmol of 6-bromocaproic acid was added, and after the reaction system was heated to 140°C, it was stirred for 12 hours. The reaction was then stopped and allowed to cool to room temperature.

The reaction mixture was filtered, and the filter cake was washed three times with 30 mL of petroleum ether. About 45 mmol of yellow solid was obtained, which was 3-(5-carboxylic acid pentyl)-2-methylbenzothiazole bromide. The yield of reaction formula I was about 78%.

NMR testing was performed on the product and the results are as follows.

1H NMR (400MHz, DMSO-d6) δ = 8.47 (d, J = 8.0Hz, 1H), 8.35 (d, J = 8.4Hz, 1H), 7.91-7.87 (m, 1H), 7.82-7.78 (m, 1H),4.73-4.70(m,2H),3.22(s,3H),2.22(t,J＝7.2Hz,2H),1.89-1.78(m,2H),1.60-1.53(m,2H),1.50 -1.41(m,2H).

In the second step, 2-((trans)-2-((trans)-6-oxo-3-((Z)-2-(5-carboxylic acid pentyl)benzothiazole- 2(3H)-yl)vinyl)cyclohex-1,4-dienyl)vinyl-3-(5-carboxylic acid pentyl)benzothiazole salt (Compound VII).

Add 1.7 mmol of 3-(5-carboxylic acid pentyl)-2-methylbenzothiazole bromide and 0.7 mmol of 4-hydroxyisophthalaldehyde obtained in the first step of the reaction to 10 mL of acetic acid, and then add 2.2 mmol sodium acetate. The reaction was stirred at a temperature of 110°C for 12 hours. After the reaction is completed, cool to room temperature and let stand for 16 hours. Afterwards, a large amount of solid precipitated, and the crude product was obtained by filtration.

The above crude product was stirred with 10 mL acetonitrile for 1 hour, and then filtered again. The filter cake was washed three times with 5 mL acetonitrile. The solid was collected and dried under reduced pressure to obtain about 0.43 mmol of brown solid powder. This brown solid powder was 2-(( trans)-2-((trans)-6-oxo-3-((Z)-2-(5-carboxylic acid pentyl)benzothiazol-2(3H)-yl)vinyl)cyclohexan-1, 4-Dienyl)vinyl-3-(5-carboxylic acid pentyl)benzothiazole salt (Compound VII), the yield of reaction formula II is about 64%.

NMR testing was performed on the product and the results are as follows.

1H NMR (400MHz, DMSO-d6) δ＝12.09-11.94(m,2H),8.76(bs,1H),8.43-8.38(m,3H),8.30(d,J＝8.4Hz,1H),8.24- 8.20(m,2H),8.16-8.10(m, 2H),7.99-7.95(m,1H),7.89-7.73(m,4H),7.02(d,J＝8.8Hz,1H),4.96-4.95(m,4H),2.23-2.18(m,4H) ,1.92-1.83(m,4H),1.62-1.47(m,8H).

After verification, the test results are consistent with the structure of structural formula 7.

Example 5 Synthesis of Compound VIII

The structure of compound VIII can be represented by structural formula 8, where Y- is an iodide ion.

In the first step, 2,3-dimethyl-5-fluorobenzothiazole quaternary ammonium iodide is prepared according to the following reaction formula I (the structure is shown on the right side of reaction formula I).

10 mL of methyl iodide was added to the reaction flask, followed by 9.0 mmol of 5-fluoro-2-methylbenzothiazole (Shanghai Haohong Biomedical Technology Co., Ltd.). The reaction was heated to 80°C and stirred at this temperature for 12 hours.

The reaction mixture was filtered, and the filter cake was washed three times with 10 mL of ethyl acetate. The filter cake was collected and dried under reduced pressure to obtain 7.1 mmol of white powder solid B. The white powdery solid is 2,3-dimethyl-5-fluorobenzothiazole quaternary ammonium iodide (the structure is shown on the right side of Reaction Formula I). The yield of Scheme I is approximately 79%.

NMR testing was performed on the product and the results are as follows.

H NMR (400MHz, DMSO-d6) δ = 8.50 (dd, J = 5.2, 9.2Hz, 1H), 8.34 (dd, J = 2.4, 8.4Hz, 1H), 7.74 (dt, J = 2.0, 8.8Hz, 1H),4.18(s,3H),3.18(s,3H).

After verification, the test results are consistent with the product structure of reaction formula I.

In the second step, prepare 5-fluoro-2-((E)-2-((E)-3-((Z)-2-(5-fluoro-3-methylbenzothiazole)) according to the following reaction formula II -2(3H)-ylidene)vinyl)-6-oxocyclohexan-1,4-dien-1-yl)ethenyl-3-methylbenzothiazolium salt (Compound VIII)

Add 2 mmol of 2,3-dimethyl-5-fluorobenzothiazole quaternary ammonium iodide (the structure is shown on the left side of reaction formula II) obtained in the first step reaction and 0.67 mmol of 4-hydroxyisophthalaldehyde. into 10mL acetic acid, then add 2.2mmol Sodium acetate. The reaction was stirred at 120°C for 12 hours.

After the reaction is completed, the reaction mixture is filtered, and the filter cake is washed three times with 10 mL acetonitrile. The solid is collected to obtain about 0.46 mmol of a brown-black solid. The brown-black solid powder is 5-fluoro-2-((E)- 2-((E)-3-((Z)-2-(5-fluoro-3-methylbenzothiazole-2(3H)-ylidene)vinyl)-6-oxocyclohexan-1, 4-Dien-1-yl)vinyl-3-methylbenzothiazolium salt (compound VIII), the yield of reaction formula II is about 95%.

NMR testing was performed on the product and the results are as follows.

1H NMR (400MHz, DMSO-d6)δ=8.72(bs,1H),8.48-8.18(m,8H),7.97-7.94(m,1H),7.73(bs,2H),7.15(bs,1H), 4.35(bs,6H).

After verification, the test results are consistent with the structure of structural formula 8.

Example 6 Synthesis of Compound IX

The structure of compound IX can be represented by structural formula 9, where Y- is an iodide ion.

In the first step, 2-methyl-5-cyanobenzothiazole is prepared according to the following reaction formula I (the structure is shown on the right side of reaction formula I).

Add 55 mL deionized water to the reaction bottle, add 0.29 mmol potassium acetate under nitrogen atmosphere and dissolve it, then add 55 mL dioxane, 5.7 mmol 5-bromo-2-methylbenzothiazole (Shanghai Bide Pharmaceutical Technology Co., Ltd. Company), 2.85mmol potassium ferricyanate trihydrate and 0.57mmol XPhos-Pd-G3. After all raw materials and reagents are added, replace with nitrogen atmosphere. The reaction mixture was reacted at 100°C for 1 hour.

After the reaction was completed, the mixture was diluted with 60 mL of water and extracted with 200 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate for 30 minutes and then filtered. The filtrate was concentrated under reduced pressure to obtain a crude product in the form of yellow oil. After the crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 10:1 to 5:1), 5.72 mmol of gray powder solid was obtained, which was 2-methyl-5-cyanobenzothiazole (structure See the right side of reaction equation I).

NMR testing was performed on the product and the results are as follows.

1H NMR (400MHz, DMSO-d6) δ = 8.44 (d, J = 1.2Hz, 1H), 8.28 (d, J = 8.6Hz, 1H), 7.79 (dd, J = 1.6, 8.4Hz, 1H), 2.85 (s,3H).

In the second step, 2,3-dimethyl-5-cyanobenzothiazole quaternary ammonium iodide is prepared according to the following reaction formula II (the structure is shown on the right side of reaction formula II).

Add 20 mL of methyl iodide to the reaction flask, followed by 5.7 mmol of 5-cyano-2-methylbenzothiazole (Chengdu Forrest Technology Development Co., Ltd.). The reaction is heated to 80°C and stirred at this temperature for 16 Hour.

The reaction mixture was filtered, and the filter cake was washed three times with 10 mL of ethyl acetate. The filter cake was collected and dried under reduced pressure to obtain 2.18 mmol of yellow powder solid, which was 2,3-dimethyl-5-cyanobenzothiazole quaternary ammonium iodide (the structure is shown on the right side of Reaction Formula II). The yield of reaction scheme II is approximately 38%.

NMR testing was performed on the product and the results are as follows.

1H NMR (400MHz, DMSO-d6) δ = 8.98 (s, 1H), 8.63 (d, J = 8.8Hz, 1H), 8.22 (dd, J = 2.0, 8.8Hz, 1H), 4.22 (s, 3H) ,3.21(s,3H).

After verification, the test results are consistent with the product structure of reaction formula II.

In the third step, 2-((trans)-2-((trans)-6-oxo-3-((Z)-2-(5-cyanobenzothiazole-2(3H) )-yl)vinyl)cyclohex-1,4-dienyl)vinyl-3-(5-cyano)benzothiazolium salt (Compound IX)

Measure 30 mL of acetic acid into a container, add 2 mmol of 5-cyano-2,3-dimethylbenzothiazole quaternary ammonium salt (the structure is shown on the left side of Reaction Formula III) and 0.67 mmol of 4 -Hydroxyisophthalaldehyde and 2.2 mmol of sodium acetate were added to the container. The reaction was stirred for 12 hours under nitrogen protection and a temperature of 120°C.

The reaction mixture was filtered, and the filter cake was washed with acetonitrile, and the filter cake was collected to obtain a crude product.

After repeatedly beating the above crude product with 10 mL of acetonitrile three times, about 0.37 mmol of brown black solid was obtained. The brown black solid powder was 2-((trans)-2-((trans)-6-oxo-3-((Z )-2-(5-cyanobenzothiazol-2(3H)-yl)vinyl)cyclohexan-1,4-dienyl)vinyl-3-(5-cyano)benzothiazolium salt ( Compound IX), the yield of reaction formula III is about 52%.

NMR testing was performed on the product and the results are as follows.

1H NMR (400MHz, DMSO-d6)δ=8.95-8.87(m,2H),8.66-8.60(m,3H),8.30-8.15(m,6H),7.93-7.89(m,1H),7.11(d ,J＝8.0Hz,1H),4.36-4.34(m,6H).

After verification, the test results are consistent with the structure of structural formula 9.

Example 7 Specificity evaluation of dyes

The fluorescence intensity change diagram of dye compound II as the concentration of calf thymus DNA and RNA increases and the linear relationship between the maximum fluorescence emission peak intensity and the concentration of calf thymus DNA and RNA are measured to evaluate the specificity of the dye.

Prepare an aqueous solution of calf thymus DNA with a certain concentration, measure its absorbance value at 260nm with a UV absorption spectrophotometer, and calibrate its concentration to 1.8mM. Take 100 μL of calf thymus DNA that has been calibrated to 1.8 mM, and add 290 μL of water to dilute the calf thymus DNA aqueous solution to 0.5 mM. Prepare a DMSO (dimethyl sulfoxide) solution of Compound B with a concentration of 1mM, take 1.5μL, add M-60LN hemolytic reagent (Mindray) to 3mL, place it in a cuvette, and measure its fluorescence intensity. Subsequently, take 0.6μL of 0.5mM calf thymus DNA aqueous solution in a cuvette each time, stir the buffer evenly, and then place it in a 37°C environment for 3 minutes to measure its fluorescence intensity. The final concentration of calf thymus DNA in the cuvette was 1 μM. Take the intensity at the maximum fluorescence emission peak of each calf thymus DNA concentration and draw a linear relationship between the fluorescence intensity and the calf thymus DNA concentration. The experiment on the linear relationship between RNA concentration and fluorescence intensity was also carried out according to the above steps. The instruments used were UV-visible spectrophotometer, model: Hp8453; fluorescence spectrophotometer, model: FP-6500.

Figure 20 shows the changes in the fluorescence spectrum of compound II as the DNA concentration increases. Figure 21 shows the changes in the fluorescence spectrum of compound II as the RNA concentration increases. Figure 22 is a linear relationship between fluorescence intensity and calf thymus DNA and RNA concentration.

It can be seen from the figure that compound II has a concentration-dependent relationship with DNA, but has no concentration-dependent relationship with RNA, indicating that compound II can specifically bind to DNA.

Example 8 Observation of staining of HepG2 living cells by Compound II under laser microscope

Add 10 μL of PBS buffer containing Compound II at a concentration of 1 mM to the six-well plate in which HepG2 cells were cultured, and incubate for 30 min in a cell culture incubator at 37°C and 5% CO2. Then, PBS was shaken and washed three times, then cell culture medium was added, and the cell morphology was observed with a confocal laser scanning microscope. Instrument model used: FV1000IX81, Japan.

Figure 23 shows the observations. The middle picture is a white field micrograph of Compound II staining HepG2 living cells. The left picture is a fluorescence micrograph of Compound II staining HepG2 living cells. The right picture is a superposition of the bright field image and the fluorescence image. As shown in the figure, it can be observed that compound II clearly stains the nucleus of HepG2 cells, indicating that the dye has good permeability and strong nucleic acid staining ability.

Example 9 Observation of staining of HepG2 living cells by Compound III under laser microscope

Add 10 μL of PBS buffer containing Compound III at a concentration of 1 mM to the six-well plate in which HepG2 cells were cultured, and incubate for 30 min in a cell culture incubator at 37°C and 5% CO2. Then, PBS was shaken and washed three times, then cell culture medium was added, and the cell morphology was observed with a confocal laser scanning microscope. Instrument model used: FV1000IX81, Japan.

Figure 24 shows the observations. The middle picture is a white field micrograph of Compound III staining HepG2 living cells. The left picture is a fluorescence micrograph of Compound III staining HepG2 living cells. The right picture is a superposition of the bright field image and the fluorescence image. As shown in the figure, it can be observed that compound III stains HepG2 cell nuclei clearly, indicating that the dye has good permeability and strong nucleic acid staining ability.

Example 10 Dye stability evaluation

Dyes used in commercial cell dyes must have certain high-temperature stability. Existing dyes have poor high-temperature stability and are subject to certain limitations in actual commercial applications. In order to improve this situation, the present invention designs and develops a variety of new dyes from the perspective of molecular structure in order to improve dye stability.

In order to evaluate the high-temperature stability, 50 mg/L of different dye/ethylene glycol solutions were placed in a 50°C thermostat, and 0.5 ml was taken at different times to test the dye concentration. The degradation curve was drawn based on the dye concentration at different time points. The dyes tested were: dye QCy-DT, dye 5 (compound V), dye 6 (compound VI), dye 9 (compound IX) reported in the literature (Nucleic Acids Research, 2015, Vol. 43, No. 18 8651-8663) ). The instrument used is UV- VIS UV-visible spectrophotometer, model: TCC-240A, SHIMADZU, Japan.

Figure 25 shows the degradation curve. The four curves from top to bottom correspond to dye 5, dye 6, dye 9, and dye QCy-DT. As can be seen from the figure, the degradation of dye QCy-DT is the most serious, while the degradation of dye 5, dye 6, and dye 9 is milder, indicating that the introduction of an electron-withdrawing group at compound R3 can improve the stability of the compound to a certain extent. , which is beneficial to commercial applications such as blood cell staining of compounds.

Example 11 Evaluation of dye cell penetration

Dyes used in commercial cell dyes need to have good cell penetration. Due to the small logP value of the molecular structure of existing dyes (the logarithmic value of the ratio of the distribution coefficient of the compound in n-octanol and water), the penetration of living cells is difficult. The permeability is weak and is subject to certain limitations in actual commercial applications. In order to improve this situation, the present invention is designed from the perspective of molecular structure and introduces a variety of chemical groups to improve the stability of the dye and at the same time enhance the penetrating power of the dye molecules into cells.

In order to verify the cell penetration ability, 10 μL of PBS buffer prepared with different dye compounds at a concentration of 1 mM was added to the 12-well plate of cultured HepG2 cells, and incubated in a cell culture incubator at 37°C and 5% CO2. Then, at different time points, the cells were shaken and washed three times with PBS, then cell culture medium was added, and a multifunctional microplate reader (Thermo, USA) was used to measure the fluorescence luminosity of each well.

Figure 26 shows the test results. The four curves correspond to dye 6, dye 9, dye 5, and dye QCy-DT from top to bottom. As shown in the figure, dyes 5, 6, and 9 have better cell penetration than dye QCy-DT.

Example 12 Use of the cyanine dye of the present invention in identifying microorganisms in blood samples to be tested using flow cytometry

First prepare dye reagent A1 and hemolytic agent B1 according to the following formula.

Wherein, the first dye includes a compound with the above structural formula 1 and is used for staining microorganisms, and the second dye includes a compound with the following chemical formula and is used for staining cells:

Then, 20 μl of dye reagent A1, 1 ml of hemolytic agent B1 and 20 μl of the anticoagulated blood sample containing Pseudomonas Klebsiella to be tested were mixed, and the mixture was allowed to dissolve at 42°C. Incubate for 30 seconds to form a sample liquid to be tested for measurement; then, use a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm to test the sample liquid to be tested to obtain the forward scattered light intensity FSC and side scatter Light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; a first scatter plot as shown in Figure 27A is generated according to the forward scattered light intensity information and the first fluorescence intensity, and according to the side scattered light intensity The information and the second fluorescence intensity generate a second scatter plot as shown in Figure 27B; based on the first scatter plot as shown in Figure 27A, it is possible to identify the presence of microorganisms in the blood sample to be tested, and based on the first scatter plot as shown in Figure 27B The two scatter plots can classify the white blood cells in the blood sample to be tested into four categories, and obtain the white blood cell classification results shown in Table 1.

The same blood sample to be tested was tested using the DIFF channel (using the DIFF supporting reagent of Mindray BC-6800) on the existing blood analyzer (Mindray, model BC-6800), and the leukocyte classification shown in Table 1 was obtained. result.

Table 1 Leukocyte classification results

As can be seen from Table 1, the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously achieve microbial detection and leukocyte detection in blood through one detection of the same blood sample to be tested.

Example 13 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples to be tested using flow cytometry

A certain amount of Pseudomonas Klebsiella was added to physiological saline to obtain a simulated body fluid sample to be tested; the above simulated body fluid sample to be tested was tested using the reagents and methods of Example 12 to obtain forward The scattered light intensity FSC and the first fluorescence intensity FL1; the first scatter plot shown in Figure 28 is generated based on the forward scattered light intensity information and the first fluorescence intensity; it can be identified based on the first scatter plot shown in Figure 28 The presence of microorganisms in simulated body fluid samples.

Example 14 Use of the cyanine dye of the present invention in identifying microorganisms in blood samples to be tested using flow cytometry

First prepare dye reagent A2 and hemolytic agent B2 according to the following formula.

Wherein, the first dye includes a compound with the above-mentioned structural formula 2 and is used for staining microorganisms, and the second dye includes a compound with the following chemical formula and is used for staining cells:

Then, 20 microliters of dye reagent A2, 1 ml of hemolytic agent B2 and 20 microliters of anticoagulated blood sample containing Pseudomonas Klebsiella to be tested were mixed, and the mixture was heated at 42°C. Incubate for 30 seconds to form a sample liquid to be tested for measurement; then, use a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm to test the sample liquid to be tested to obtain the forward scattered light intensity FSC and side scattering Light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; a first scatter plot as shown in Figure 29A is generated according to the forward scattered light intensity information and the first fluorescence intensity, and according to the side scattered light intensity The information and the second fluorescence intensity generate a second scatter plot as shown in Figure 29B; based on the first scatter plot as shown in Figure 29A, it is possible to identify the presence of microorganisms in the blood sample to be tested, and based on the first scatter plot as shown in Figure 29B The two scatter plots can classify the white blood cells in the blood sample to be tested into four categories, and obtain the white blood cell classification results shown in Table 2.

The same blood sample to be tested was tested using the DIFF channel (using Mindray BC-6800's DIFF supporting reagent) on the existing blood analyzer (Mindray, model BC-6800), and the leukocyte classification shown in Table 2 was obtained. result.

Table 2 Leukocyte classification results

As can be seen from Table 2, the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously achieve microbial detection and leukocyte detection in blood through one detection of the same blood sample to be tested.

Example 15 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples to be tested using flow cytometry

A certain amount of Pseudomonas Klebsiella was added to the physiological saline to obtain a simulated body fluid sample to be tested; the above simulated body fluid sample to be tested was tested using the reagents and methods of Example 14 to obtain forward The scattered light intensity FSC and the first fluorescence intensity FL1; the first scatter plot shown in Figure 30 is generated based on the forward scattered light intensity information and the first fluorescence intensity; it can be identified based on the first scatter plot shown in Figure 11 The presence of microorganisms in simulated body fluid samples.

Example 16 Use of the cyanine dye of the present invention in identifying microorganisms in blood samples to be tested using flow cytometry

First prepare dye reagent A3 and hemolytic agent B3 according to the following formula.

Wherein, the first dye includes a compound with the above-mentioned structural formula 3 and is used for staining microorganisms, and the second dye includes a compound with the following chemical formula and is used for staining cells:

Then, 20 μl of dye reagent A3, 1 ml of hemolytic agent B3 were mixed with 20 μl of anticoagulated blood sample containing E. coli to be tested, and the mixture was incubated at 42°C for 30 seconds to form the assay. The sample liquid to be tested is tested; then, a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm is used to test the sample liquid to be tested to obtain the forward scattered light intensity FSC, side scattered light intensity SSC, and the first Fluorescence intensity FL1 and second fluorescence intensity FL2; generate a first scatter plot as shown in Figure 31A based on the forward scattered light intensity information and the first fluorescence intensity, and generate a first scatter plot as shown in Figure 31A based on the side scattered light intensity information and the second fluorescence intensity A second scatter plot as shown in FIG. 31B is generated; based on the first scatter plot as shown in FIG. 31A, it can be identified that the presence of microorganisms in the blood sample to be tested is detected, and based on the second scatter plot as shown in FIG. 31B, it can be treated The white blood cells in the blood sample were measured and classified into four categories, and the white blood cell classification results were obtained as shown in Table 3.

The same blood sample to be tested was tested using the DIFF channel (using Mindray BC-6800's DIFF supporting reagent) on the existing blood analyzer (Mindray, model BC-6800), and the leukocyte classification shown in Table 3 was obtained. result.

Table 3 White blood cell classification results

It can be seen from Table 3 that the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously achieve microbial detection and leukocyte detection in blood through one detection of the same blood sample to be tested.

Example 17 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples to be tested using flow cytometry

A certain amount of Escherichia coli is added to the physiological saline to obtain a simulated body fluid sample to be tested; the above simulated body fluid sample to be tested is tested using the reagents and methods of Example 16 to obtain the forward scattered light intensity FSC and the third 1. Fluorescence intensity FL1; generate a first scatter diagram as shown in Figure 32 based on the forward scattered light intensity information and the first fluorescence intensity; based on the first scatter diagram shown in Figure 32, it can be identified that in the simulated body fluid sample Microorganisms are present.

Example 18 Use of the cyanine dye of the present invention in identifying microorganisms in blood samples to be tested using flow cytometry

First prepare dye reagent A4 and hemolytic agent B4 according to the following formula.

Then, 20 μl of dye reagent A4, 1 ml of hemolytic agent B4 and 20 μl of the anticoagulated blood sample containing Pseudomonas Klebsiella to be tested were mixed, and the mixture was allowed to dissolve at 42°C. Incubate for 30 seconds to form a sample liquid to be tested for measurement; then, use a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm to test the sample liquid to be tested to obtain the forward scattered light intensity FSC, first fluorescence Intensity FL1 and second fluorescence intensity FL2; generate a first scatter plot as shown in Figure 33A based on the forward scattered light intensity information and the first fluorescence intensity, and generate based on the forward scattered light intensity information and the second fluorescence intensity As shown in the second scatter plot shown in Figure 33B; based on the first scatter plot shown in Figure 33A, it can be identified that the microorganism is present in the blood sample to be tested, and based on the second scatter plot shown in Figure 33B, the presence of microorganisms in the blood sample to be tested can be identified. A blood sample is tested and counted for white blood cells, nucleated red blood cells, and basophils.

It can be seen that the embodiments of the present invention can simultaneously realize the detection of microorganisms in the blood and the detection of nucleated red blood cells through one detection of the same blood sample to be tested.

Example 19 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples to be tested using flow cytometry

Add a certain amount of Pseudomonas Klebsiella to physiological saline to obtain a simulated body fluid sample to be tested; collect The above simulated body fluid sample to be tested was tested using the reagents and methods of Example 18 to obtain the forward scattered light intensity FSC and the first fluorescence intensity FL1; according to the forward scattered light intensity information and the first fluorescence intensity, the following figure is generated: The first scatter plot shown in Figure 34; Based on the first scatter plot shown in Figure 34, it can be identified that microorganisms are present in the simulated body fluid sample.

Example 20 Use of the cyanine dye of the present invention in identifying microorganisms in blood samples to be tested using flow cytometry

First prepare dye reagent A5 and hemolytic agent B5 according to the following formula.

Wherein, the first dye includes a compound with the above structural formula 3 and is used for staining microorganisms, and the second dye includes a compound with the following chemical formula and is used for staining cells:

Then, 20 microliters of dye reagent A5, 1 ml of hemolytic agent B5 and 20 microliters of anticoagulated blood sample containing Pseudomonas Klebsiella to be tested were mixed, and the mixture was heated at 42°C. Incubate for 30 seconds to form a sample liquid to be tested for measurement; then, use a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm to test the sample liquid to be tested to obtain the forward scattered light intensity FSC, first fluorescence Intensity FL1 and second fluorescence intensity FL2; root The first scatter plot shown in Figure 35A is generated based on the forward scattered light intensity information and the first fluorescence intensity, and the second scatter plot shown in Figure 35B is generated based on the forward scattered light intensity information and the second fluorescence intensity. Dot plot; the presence of microorganisms in the blood sample to be tested can be identified based on the first scatter plot shown in Figure 35A, and the leukocytes, nucleated cells in the blood sample to be tested can be identified based on the second scatter plot shown in Figure 35B red blood cells and basophils and count them.

Example 21 Use of the cyanine dye of the present invention in identifying microorganisms in simulated body fluid samples to be tested using flow cytometry

A certain amount of Pseudomonas Klebsiella was added to the physiological saline to obtain a simulated body fluid sample to be tested; the above simulated body fluid sample to be tested was tested using the reagents and methods of Example 20 to obtain forward The scattered light intensity FSC and the first fluorescence intensity FL1; the first scatter plot shown in Figure 36 is generated according to the forward scattered light intensity information and the first fluorescence intensity; it can be identified based on the first scatter plot shown in Figure 36 The presence of microorganisms in simulated body fluid samples.

Example 22 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry

20 microliters of dye reagent A4 (Example 18, the first dye is used to stain parasites), 1 ml of hemolytic agent B4 (Example 18) and 20 microliters of anticoagulated malaria-infected The patient's blood sample to be tested is mixed, and the mixture is incubated at 42°C for 30 seconds to form a sample liquid to be tested; then, a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm is used to test the sample liquid Conduct a test to obtain the forward scattered light intensity FSC, the first fluorescence intensity FL1 and the second fluorescence intensity FL2; generate a first scatter plot as shown in Figure 37A based on the forward scattered light intensity information and the first fluorescence intensity, And generate a second scatter plot as shown in Figure 37B based on the forward scattered light intensity information and the second fluorescence intensity; based on the first scatter plot as shown in Figure 37A, it can be identified that malaria parasites are present in the blood sample to be tested , and the white blood cells, nucleated red blood cells and basophils in the blood sample to be tested can be identified and counted based on the second scatter plot shown in FIG. 37B.

It can be seen that embodiments of the present invention can simultaneously detect parasites in blood and detect nucleated red blood cells through one detection of the same blood sample to be tested.

Example 23 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry

First prepare dye reagent A6 and hemolytic agent B6 according to the following formula.

Wherein, the first dye includes a compound with the above-mentioned structural formula 2 and is used for staining parasites, and the second dye includes a compound with the following chemical formula and is used for staining cells:

Then, 20 μl of dye reagent A6, 1 ml of hemolytic agent B6 were mixed with 20 μl of the anticoagulated blood sample to be tested from a patient infected with malaria, and the mixture was incubated at 42°C for 30 seconds. Form a sample liquid to be tested for measurement; then, use a flow cytometer with a blue laser with an excitation wavelength of about 450 nm to test the sample liquid to be tested to obtain the forward scattered light intensity FSC, the first fluorescence intensity FL1 and the third 2. Fluorescence intensity FL2; generate a first scatter plot as shown in Figure 38A based on the forward scattered light intensity information and the first fluorescence intensity, and generate a first scatter plot as shown in Figure 38B based on the forward scattered light intensity information and the second fluorescence intensity. The second scatter plot shown in Figure 38A can identify the presence of malaria parasites in the blood sample to be tested based on the first scatter plot shown in Figure 38A, and the blood sample to be tested can be identified based on the second scatter plot shown in Figure 38B white blood cells, nucleated red blood cells, and basophils and count them.

Example 24 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry

20 microliters of dye reagent A5 (Example 20, the first dye is used to stain parasites), 1 ml of hemolytic agent B5 (Example 20) and 20 microliters of anticoagulated malaria-infected The patient's blood sample to be tested is mixed, and the mixture is incubated at 42°C for 30 seconds to form a sample liquid to be tested; then, a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm is used to test the sample liquid Conduct a test to obtain the forward scattered light intensity FSC, the first fluorescence intensity FL1 and the second fluorescence intensity FL2; generate a first scatter plot as shown in Figure 39A based on the forward scattered light intensity information and the first fluorescence intensity, And generate a second scatter plot as shown in Figure 39B based on the forward scattered light intensity information and the second fluorescence intensity; based on the first scatter plot as shown in Figure 39A, it can be identified that malaria parasites are present in the blood sample to be tested , and the white blood cells, nucleated red blood cells and basophils in the blood sample to be tested can be identified and counted based on the second scatter plot shown in FIG. 39B.

Example 25 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry

20 microliters of dye reagent A2 (Example 14, the first dye is used to stain parasites), 1 ml of hemolytic agent B2 (Example 14) and 20 microliters of anticoagulated malaria-infected The patient's blood sample to be tested is mixed, and the mixture is incubated at 42°C for 30 seconds to form a sample liquid to be tested; then, a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm is used to test the sample liquid Carry out testing to obtain the forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; generate according to the forward scattered light intensity information and the first fluorescence intensity as shown in Figure 40A The first scatter plot, and the second scatter plot shown in Figure 40B is generated based on the side scattered light intensity information and the second fluorescence intensity; based on the first scatter plot shown in Figure 40A, it can be identified that the patient is to be Plasmodium is present in the blood sample to be tested, and the white blood cells in the blood sample to be tested can be classified into four categories based on the second scatter plot shown in Figure 40B, and the white blood cell classification results shown in Table 4 are obtained.

Use the DIFF channel (using Mindray BC-6800's DIFF supporting reagent) on the existing blood analyzer (Mindray, model BC-6800) to test the same blood sample to be tested, and obtain the leukocyte classification shown in Table 4 result.

Table 4 Leukocyte classification results

It can be seen from Table 4 that the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously detect parasites and white blood cells in blood through one detection of the same blood sample to be tested.

Example 26 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry

First prepare dye reagent A7 and hemolytic agent B7 according to the following formula.

Wherein, the first dye includes a compound with the structural formula 3 in the above Table 1 and is used for staining parasites, and the second dye includes a compound with the following chemical formula and is used for staining cells:

Then, 20 μl of dye reagent A7, 1 ml of hemolytic agent B7 were mixed with 20 μl of the anticoagulated blood sample to be tested from a malaria-infected patient, and the mixture was incubated at 42°C for 30 seconds. A sample liquid to be tested is formed for measurement; then, a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm is used to test the sample liquid to be tested to obtain the forward scattered light intensity FSC, side scattered light intensity SSC, The first fluorescence intensity FL1 and the second fluorescence intensity FL2; generate a first scatter plot as shown in Figure 41A according to the forward scattered light intensity information and the first fluorescence intensity, and generate a first scatter plot as shown in Figure 41A according to the side scattered light intensity information and the second The fluorescence intensity generates a second scatter plot as shown in Figure 41B; based on the first scatter plot as shown in Figure 41A it is possible to identify the presence of Plasmodium in the blood sample to be tested, and based on the second scatter plot as shown in Figure 41B The graph can classify the white blood cells in the blood sample to be tested into four categories, and obtain the white blood cell classification results shown in Table 5.

The same blood sample to be tested was tested using the DIFF channel (using Mindray BC-6800's DIFF supporting reagent) on the existing blood analyzer (Mindray, model BC-6800), and the leukocyte classification shown in Table 5 was obtained. result.

Table 5 Leukocyte classification results

It can be seen from Table 5 that the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously detect parasites and white blood cells in blood through one detection of the same blood sample to be tested.

Example 27 Use of the cyanine dye of the present invention in identifying parasites in blood samples to be tested using flow cytometry

First prepare dye reagent A8 and hemolytic agent B8 according to the following formula.

Wherein, the first dye includes a compound with the above-mentioned structural formula 4 and is used for staining parasites, and the second dye includes a compound with the following chemical formula and is used for staining cells:

Then, 20 μl of dye reagent A8, 1 ml of hemolytic agent B8 were mixed with 20 μl of the anticoagulated blood sample to be tested from a malaria-infected patient, and the mixture was incubated at 42°C for 30 seconds. A sample liquid to be tested is formed for measurement; then, a flow cytometer with a blue laser with an excitation wavelength of approximately 450 nm is used to test the sample liquid to be tested to obtain the forward scattered light intensity FSC, side scattered light intensity SSC, The first fluorescence intensity FL1 and the second fluorescence intensity FL2; generate a first scatter plot as shown in Figure 42A according to the forward scattered light intensity information and the first fluorescence intensity, and generate a first scatter plot as shown in Figure 42A according to the side scattered light intensity information and the second The fluorescence intensity generates a second scatter plot as shown in Figure 42B; based on the first scatter plot as shown in Figure 42A, the presence of malaria parasites in the blood sample to be tested can be identified, and based on the second scatter plot as shown in Figure 42B The graph can classify the white blood cells in the blood sample to be tested into four categories, and obtain the white blood cell classification results shown in Table 6.

Use the DIFF channel (using Mindray BC-6800's DIFF supporting reagent) on the existing blood analyzer (Mindray, model BC-6800) to test the same blood sample to be tested, and obtain the leukocyte classification shown in Table 6 result.

Table 6 Leukocyte classification results

It can be seen from Table 6 that the leukocyte classification results obtained according to the present invention are basically consistent with the leukocyte classification results obtained according to the existing BC-6800. It can be seen that embodiments of the present invention can simultaneously detect parasites and white blood cells in blood through one detection of the same blood sample to be tested.

The features or combinations of features mentioned above in the description, drawings and claims can be used in any combination with each other or alone as long as they are meaningful within the scope of the invention and do not conflict with each other. The advantages and features described with respect to the sample analysis method provided by the invention apply in a corresponding manner to the use of the sample analyzer and DNA-specific dyes provided by the invention, and vice versa.

The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. Under the inventive concept of the present invention, equivalent transformations can be made by using the contents of the description and drawings of the present invention, or directly/indirectly applied in Other related technical fields are included in the patent protection scope of the present invention.

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all teachings that have been published, and these changes are within the protection scope of the present invention. . The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

A compound having a structure as shown in general formula I or its hydrate, solvate, stereoisomer, tautomer or crystal form,

in,

R1 and R2 are the same or different, and are independently selected from C1-18 linear or branched alkyl, C1-18 linear or branched alkylene-M, M is selected from sulfonic acid group, phenyl, carboxyl, mercapto group , amino;

R3 is selected from hydrogen, sulfonic acid group, halogen, cyano group, C1-6 alkyl group, hydroxyl group, C1-6 alkoxy group, and halogenated C1-6 alkyl group;

Y does not exist or is a counter anion;

And limited to:

When R3 is hydrogen, R1 and R2 are not both methyl and R1 and R2 are not both benzyl.
The compound of claim 1 or its hydrate, solvate, stereoisomer, tautomer or crystal form, wherein R1 and R2 are the same or different, and are independently selected from C1-6 linear alkane group, C1-6 linear alkylene group -M, M is selected from the group consisting of sulfonic acid group, phenyl group, carboxyl group, mercapto group and amino group.
The compound of claim 1 or 2 or its hydrate, solvate, stereoisomer, tautomer or crystal form, wherein at least one of R1 and R2 is C1-18 linear or branched chain Alkylene-sulfonic acid group.
The compound of any one of claims 1-3 or its hydrate, solvate, stereoisomer, tautomer or crystal form, wherein R1 and R2 are different and independently selected from C1-6 straight Alkyl group, benzyl group, C1-6 linear alkylene-carboxy group, C1-6 linear alkylene-sulfonic acid group, C1-6 linear alkylene-mercapto group, C1-6 linear alkylene group -Amino.
The compound of any one of claims 1-3 or its hydrate, solvate, stereoisomer, tautomer or crystal form, wherein R1 and R2 are the same and selected from C1-6 linear alkane group, C1-6 linear alkylene-sulfonic acid group, C1-6 linear alkylene-carboxyl group.
The compound or its hydrate, solvate, stereoisomer, tautomer or crystal form according to any one of claims 1 to 5, wherein R3 is selected from hydrogen, sulfonic acid group, halogen, cyano group , C1-6 alkyl.
The compound or its hydrate, solvate, stereoisomer, tautomer or crystal form according to any one of claims 1 to 6, wherein when Y is a counter anion, Y is selected from the group consisting of halide ions ( For example, F-, Cl-, Br-, I-), ClO4-, PF6-, CF3SO3-, BF4-, acetate, methanesulfonate or p-toluenesulfonate.
The compound or its hydrate, solvate, stereoisomer, tautomer or crystal form according to any one of claims 1 to 7, wherein when Y is absent, the compound is an internal salt.
The compound of claim 8 or its hydrate, solvate, stereoisomer, tautomer or crystal form, wherein at least one of R1 and R2 is selected from C1-18 linear or branched chain subunits. Alkyl-sulfonic acid group, C1-18 linear or branched alkylene-carboxylic group.
The compound, its hydrate, solvate, stereoisomer, tautomer or crystal form according to any one of claims 1 to 9, the compound has any of the following structures:
The compound or its hydrate, solvate, stereoisomer, tautomer or crystal form according to any one of claims 1 to 9, the compound has any of the following structures:
A conjugate comprising a compound of any one of claims 1-11 or a hydrate, solvate, stereoisomer, tautomer or crystalline form thereof.
A composition for staining biological samples, wherein the composition comprises the compound of any one of claims 1-11 or its hydrate, solvate, stereoisomer, tautomer or crystal form or The conjugate of claim 12.
The composition of claim 13, wherein the biological sample is nucleic acid, preferably deoxyribonucleic acid.
The compound of any one of claims 1 to 11 or its hydrate, solvate, stereoisomer, tautomer or crystal form, the conjugate of claim 12 or the composition of claim 13 in biological Use in staining samples.
The use of claim 15, wherein the biological sample is nucleic acid, preferably deoxyribonucleic acid.
The compound of any one of claims 1 to 11 or its hydrate, solvate, stereoisomer, tautomer or crystal form, the conjugate of claim 12 or the composition of claim 13 is used in the process Cytometry is used to identify parasites in blood samples to be tested.
The compound of any one of claims 1 to 11 or its hydrate, solvate, stereoisomer, tautomer or crystal form, the conjugate of claim 12 or the composition of claim 13 is used in the process Cytometry is used to identify microorganisms in blood samples to be tested or body fluid samples to be tested.
A sample analysis method, characterized in that the sample analysis method includes the following steps:

Using a dye reagent containing the first dye and a hemolytic agent for lysing red blood cells to process the same biological sample to be tested to obtain a sample liquid to be tested, wherein the biological sample to be tested is a blood sample to be tested or a body fluid sample to be tested, The first dye can dye microorganisms;

Make the particles in the sample liquid to be tested pass through the optical detection area one by one and irradiate the particles flowing through the optical detection area with light to obtain the scattered light information generated by the particles in the sample liquid to be tested after being irradiated with light. Fluorescence information including first fluorescence information from the first dye; and

Microorganisms in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.
A sample analyzer, characterized in that the sample analyzer includes:

A sampling device for quantitatively absorbing biological samples to be tested, wherein the biological samples to be tested are blood samples to be tested or body fluid samples to be tested;

A sample preparation device having a reaction tank and a reagent supply part, wherein the reaction tank is used to receive the biological sample to be tested drawn by the sampling device, and to receive a dye containing a first dye provided by the reagent supply part Reagents and hemolytic reagents for lysing red blood cells. The biological sample to be tested absorbed by the sampling device is mixed with the dye reagent and hemolytic reagent provided by the reagent supply part in the reaction tank to prepare a sample to be tested. Liquid, wherein the first dye can dye microorganisms;

Optical detection device, including a light source, a flow chamber, a scattered light detector and a fluorescence detector. The light source is used to emit a light beam to illuminate the flow chamber. The flow chamber is connected to the reaction cell and the sample liquid to be tested is The particles in the flow chamber can pass through the flow chamber one by one, the scattered light detector is used to detect the scattered light information generated by the particles passing through the flow chamber after being irradiated by light, and the fluorescence detector is used to detect the particles passing through the flow chamber. Fluorescence information generated by the particles after being illuminated by light, the fluorescence information including first fluorescence information from the first dye; and

A processor configured to acquire the scattered light information and the fluorescence information from the optical detection device, and identify microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information.
The sample analyzer of claim 20, wherein the processor is further configured to perform the following when identifying microorganisms in the sample liquid to be tested based on the scattered light information and the first fluorescence information. step:

Generate a first scatter plot based on the scattered light information, preferably forward scattered light information, and the first fluorescence information; and

Microorganisms in the sample fluid to be tested are identified based on the first scatter plot.
The sample analyzer of claim 21, wherein the processor is further configured to:

A microbial characteristic area and a leukocyte area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the microbial characteristic area is greater than the intensity of the first fluorescence information of the leukocyte area; and

Microorganisms in the sample fluid to be tested are identified based on the microorganism characteristic areas.
The sample analyzer according to any one of claims 20 to 22, characterized in that the first dye is a dye that can specifically bind to deoxyribonucleic acid, especially the first dye includes a dye as claimed in claim 1 The compound described in any one of -11.
A blood analysis method, characterized in that the blood analysis method includes the following steps:

Using a dye reagent containing a first dye and a hemolytic agent for lysing red blood cells to process the same blood sample to be tested to obtain a sample liquid to be tested, wherein the first dye can stain parasites in the blood, and the The first dye and the first dye include a compound having a structure of general formula I, especially a compound as described in any one of claims 1-11;

Wherein, R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, and M is selected from sulfonic acid group, Phenyl, carboxyl, mercapto, amino, R 3 is selected from hydrogen, sulfonic acid group, halogen, cyano, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy, halogenated C 1-6 alkyl, Y does not exist or is a counter anion;

Make the particles in the sample liquid to be tested pass through the optical detection area one by one and irradiate the particles flowing through the optical detection area with light to obtain the scattered light information generated by the particles in the sample liquid to be tested after being irradiated with light. Fluorescence information including first fluorescence information from the first dye; and

Parasites in the sample fluid to be tested are identified based on the scattered light information and the first fluorescence information.
A blood analyzer, characterized in that the blood analyzer includes:

Sampling device, used to quantitatively draw blood samples to be tested;

A sample preparation device having a reaction tank and a reagent supply part, wherein the reaction tank is used to receive the blood sample to be tested drawn by the sampling device, and to receive a dye containing a first dye provided by the reagent supply part Reagents and hemolytic reagents for lysing red blood cells. The blood sample to be tested drawn by the sampling device is mixed with the dye reagent and hemolytic reagent provided by the reagent supply part in the reaction tank to prepare a sample to be tested. Liquid, wherein the first dye includes a compound having a structure of general formula I, especially a compound as described in any one of claims 1-11;

Wherein, R 1 and R 2 are the same or different, and are independently selected from C 1-18 linear or branched alkyl, C 1-18 linear or branched alkylene-M, and M is selected from sulfonic acid group, Phenyl, carboxyl, mercapto, amino, R 3 is selected from hydrogen, sulfonic acid group, halogen, cyano, C 1-6 alkyl, hydroxyl, C 1-6 alkoxy, halogenated C 1-6 alkyl, Y does not exist or is a counter anion;

Optical detection device, including a light source, a flow chamber, a scattered light detector and a fluorescence detector. The light source is used to emit a light beam to illuminate the flow chamber. The flow chamber is connected to the reaction cell and the sample liquid to be tested is The particles in the flow chamber can pass through the flow chamber one by one, the scattered light detector is used to detect the scattered light information generated by the particles passing through the flow chamber after being irradiated by light, and the fluorescence detector is used to detect the particles passing through the flow chamber. Fluorescence information generated by the particles after being illuminated by light, the fluorescence information including first fluorescence information from the first dye; and

A processor configured to acquire the scattered light information and the fluorescence information from the optical detection device, and identify parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information.
The blood analyzer of claim 25, wherein the processor is further configured to execute when identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information. Following steps:

Generate a first scatter plot based on the scattered light information, preferably forward scattered light information, and the first fluorescence information; and

Parasites in the sample fluid to be tested are identified based on the first scatter plot.
The blood analyzer of claim 26, wherein the processor is further configured to:

A parasite characteristic area and a leukocyte area are obtained from the first scatter plot, and the intensity of the first fluorescence information of the parasite characteristic area is greater than the intensity of the first fluorescence information of the leukocyte area; and

Parasites in the sample fluid to be tested are identified based on the parasite characteristic area.