A non-washing two-color cell imaging fluorescein carbon dot staining solution for fungal detection, staining method and application

Abstract

The present invention discloses a non-washing two-color cell imaging fluorescein carbon dot staining solution for fungal detection, which, by mass percentage, comprises: 0.50% - 1.50% of fluorescein carbon dot material and 98.50% - 99.50% of water; wherein the fluorescein carbon dot material is made from raw materials with the following mass percentages: 0.5% - 1.3% of fluorescent brightener 135#, 25% - 29% of ethylenediamine, 0.05% - 0.13% of ammonium persulfate, and 69.57% - 74.45% of water. Also disclosed is a method for fungal staining using the above staining solution and the application of the above fluorescein carbon dot staining solution in fungal detection using fluorescence staining method. In particular, this staining solution can accurately determine the presence of epidermal fungi without removing dandruff, has simple components, can remain stable for a long time, and greatly reduces the cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescein carbon dot staining solutions, and particularly relates to a washing-free two-color cell imaging fluorescein carbon dot staining solution for fungal detection, a staining method and applications thereof. Background Art

[0002] The detection of epidermal fungi has an important guiding role in the diagnosis and prevention of skin and hair diseases. Traditional methods for detecting epidermal fungi mainly include fungal smear microscopy and fungal culture. For smear microscopy, the specimen taken is directly placed on a glass slide, stained or added with 10% KOH solution, and examined under a light microscope. However, the contrast between fungi and tissue cells is not significant, and it is very difficult to identify fungi. To a large extent, it depends on the experience of the operator to interpret the results, and there is a great possibility of misdetection. Fungal culture is the gold standard for the diagnosis of superficial fungal infections. However, due to the complexity of pathogenic bacteria, it takes a long time and is difficult to meet the clinical requirements in actual daily work.

[0003] In recent years, as an improved technology of the traditional fungal microscopy method, the fungal fluorescence staining method has received more and more extensive attention in clinical practice due to its rapidity, sensitivity and high specificity. By using a fluorescent dye, under the irradiation of a specific excitation light band of a fluorescence microscope, the fungi targeted by the fluorescent dye can form a significant contrast with the dark background, making the morphological structure of the fungi clearly visible under the microscope and easy to observe, greatly improving the work efficiency and the sensitivity of clinical fungal detection. Currently, the fluorescent reagent formulations used in epidermal fungal detection on the market are complex, cumbersome to prepare and have unstable performance. Fluorescent reagents generally include three main components: a fluorescent brightener, potassium hydroxide and a background counterstain: 1) The fluorescent brightener combines with the β-polysaccharide component in the tissue to produce fluorescence; 2) Potassium hydroxide can dissolve the cutin in the sample tissue, eliminate impurities and make the tissue transparent; 3) The background counterstain can reduce the background brightness of the sample tissue and make the fluorescence signal clearer. However, due to the poor stability of the fluorescent brightener in a strong alkaline environment, precipitation of fluorescein will occur; moreover, the background counterstain will also affect the stability of the fluorescent brightener, leading to a decrease in detection sensitivity; and the staining steps are cumbersome and cannot meet the clinical requirements for rapid screening of fungi. Therefore, it is necessary to provide a fungal detection method that does not require the combination of other complex reagents and has stable performance to solve the deficiencies in the prior art.

[0004] Carbon dots refer to zero-dimensional carbon-based nanoparticles with at least one dimension size in the range of 10 nm. In recent years, due to their excellent biocompatibility, photoluminescence and other properties, they have attracted much attention and have been widely used in fields such as chemical sensing, drug delivery, photocatalysts and food detection. Bioimaging is also one of the most widely used fields. It has been experimentally verified that the strategy of targeting microorganisms by modifying various affinity groups on the carbon core surface can effectively meet the clinical requirements for rapid screening of microorganisms.

[0005] One of the prior art fluorescent stains, which is involved in the detection of fungi and skin parasites, has a complex composition and is composed of water, fluorescent brightener, potassium hydroxide, background counterstain, solubilizer and humectant.

[0006] It has the following disadvantages: 1) With a complex composition, generally a background counterstain needs to be added to reduce the background brightness of the sample tissue, and alkalis are required to dissolve the cutin in the sample tissue and eliminate impurities, so as to make the fluorescence signal clearer. However, these solvents will affect the stability of the fluorescent brightener, resulting in unstable performance, and other reagents are needed to maintain stability, leading to cumbersome solution preparation; 2) After staining, the fungi show blue fluorescence under a fluorescence microscope, which is harmful to the eyes; 3) The biocompatibility of the fluorescent brightener, alkali, etc. is poor, and there is certain biological toxicity.

[0007] The fluorescent staining solution of the second prior art has a composition including wheat germ agglutinin, fluorescein isothiocyanate, propidium iodide, sodium dihydrogen phosphate, disodium hydrogen phosphate, glycerol, Proclin 300 and water, and its function is to combine with morphology to detect microorganisms in vaginal secretion samples, cervical exfoliated cell samples, skin samples and sputum samples.

[0008] It has the following disadvantages: 1) This staining solution uses the specific recognition of lectin for glycoprotein and the specific staining of nucleic acid dye for nucleic acid, with high cost; 2) The composition is complex, the preparation is cumbersome, and the performance of the fluorescein is unstable; 3) High requirements for slide preparation and cumbersome steps.

[0009] The fluorescent staining solution of the third prior art also contains a fluorescent carbon dot material, and the raw materials are fluorescent brightener 33, citric acid and ethylenediamine. It has the following disadvantages: It can observe fungi cultured in the laboratory, but does not involve the detection of fungi without eliminating the influence of impurities such as dandruff, and cannot meet the requirements of clinical rapid screening. Summary of the Invention

[0010] In order to solve the problems of the current fluorescent staining solution with complex composition, unstable performance and single luminescence, the first object of the present invention is to provide a washing-free two-color cell imaging fluorescent carbon dot staining solution for the detection of fungi, especially dermatophytes. This staining solution only contains fluorescent carbon dots and water, has a simple composition, stable performance, can perform two-color imaging, can make the sample to be tested washing-free, and does not quench under a fluorescence microscope for a long time.

[0011] The present invention also aims to provide a method for staining fungi, especially dermatophytes, using the above staining solution. This method is simple and easy to operate, time-consuming, and the staining solution has good targeting ability for bacteria, can clearly distinguish dandruff and bacteria through morphology, and has high quality of the formed film.

[0012] The last object of the present invention is to provide the application of the above-mentioned fluorescein carbon dot staining solution in the detection of fungi, especially dermatophytes, by fluorescence staining method.

[0013] The first object of the present invention can be achieved by the following technical solution: A two-color cell imaging fluorescein carbon dot staining solution for fungal detection, by mass percentage, includes: 0.50% - 1.50% of fluorescein carbon dot material, 98.50% - 99.50% of water; wherein the fluorescein carbon dot material is made from raw materials with the following mass percentages: 0.5% - 1.3% of fluorescent brightener 135#, 25% - 29% of ethylenediamine, 0.05% - 0.13% of ammonium persulfate, 69.57% - 74.45% of water.

[0014] Fluorescent brightener 135# is a fluorescein that emits blue light under the irradiation of excitation light and is slightly soluble in water. During microscopic examination, in order to obtain better imaging, strong alkali is often used to dissolve tissues. However, the addition of alkali will greatly affect the stability of the fluorescent brightener, resulting in unstable performance. Therefore, in this application, carbon dots are first formed by one-step hydrothermal carbonization using fluorescent brightener 135#, ethylenediamine, ammonium persulfate, and water as raw materials. Among them, ethylenediamine with good reducibility helps the carbon dots nucleate, and the oxidizing property of ammonium persulfate further improves the optical properties of the carbon dots. The molecular structure of fluorescent brightener 135# is smaller and more likely to nucleate, thus a fluorescein carbon dot material with good water solubility, stability, yellow light emission, and strong targeting ability to fungi is prepared.

[0015] The raw materials for preparing the fluorescein carbon dot material of the present invention are: ethylenediamine, fluorescent brightener 135#, ammonium persulfate, and water.

[0016] The fluorescein carbon dot material of the present invention is preferably prepared by the following method: Select fluorescent brightener 135#, ammonium persulfate, ethylenediamine, and water, and prepare it by one-step hydrothermal reaction. The reaction product is filtered to obtain the fluorescein carbon dot material.

[0017] Preferably, the fluorescein carbon dot material is prepared by one-step hydrothermal reaction, including carrying out hydrothermal reaction at a temperature of 120 - 180°C for 8 - 12 hours.

[0018] More preferably, the fluorescein carbon dot material is prepared by one-step hydrothermal reaction, including carrying out hydrothermal reaction in an oven at a temperature of 140°C for 10 hours.

[0019] The second object of the present invention can be achieved by the following technical solution: A method for fungal staining using the above-mentioned fluorescein carbon dot staining solution, including the following steps:

[0020] 1) Slide preparation: Take a clean glass slide, drop water in the middle area, transfer the sample to be tested to the water drop, heat and dry it, and the slide preparation is completed;

[0021] 2) Staining: Drop the fluorescein carbon dot staining solution onto the sample to be tested, cover it, and stain for 2 min to 10 min, then air dry.

[0022] 3) Sealing the slide: Take a cover slip and directly cover the stained slide.

[0023] 4) Microscopic examination: Take the sample slide and observe it under a fluorescence microscope in the UV and blue light bands to check the morphology and fluorescence intensity of the fungi in the sample to be tested.

[0024] Preferably, in step 4), the observation is carried out under ultraviolet light in the wavelength band of 320 - 400 nm. The sample to be tested shows cyan fluorescence under ultraviolet excitation. The observation is carried out under blue light in the wavelength band of 400 - 470 nm, and the sample to be tested shows bright yellow fluorescence under blue light excitation.

[0025] More preferably, in step 4), the observation is carried out under ultraviolet light at 365 nm. The sample to be tested shows cyan fluorescence under ultraviolet excitation. The observation is carried out under blue light at 455 nm, and the sample to be tested shows bright yellow fluorescence under blue light excitation.

[0026] The above last object of the present invention can be achieved by the following technical solution: The application of the above fluorescein carbon dot staining solution in the detection of fungi, especially dermatophytes, by fluorescence staining method.

[0027] The present invention has the following advantages:

[0028] (1) The preparation of the fluorescein carbon dot material in the present invention is simple. It can be prepared in one step by the hydrothermal method. The formula of the present invention is a single dosage form, with simple components and good product stability.

[0029] (2) The preparation of the staining solution in the present invention is simple and fast. Staining can be completed with only one operation.

[0030] (3) The fluorescence staining solution of the present application can not only label the fungi cultured in the laboratory, but also immediately detect dermatophytes without strong alkali and solubilizer, fully meeting the requirements of rapid screening in clinical practice, and the staining solution formula has low biological toxicity.

[0031] (4) The present invention provides carbon dots (CDs) synthesized by a hydrothermal reaction method using fluorescent brightener 135#, ethylenediamine, and ammonium persulfate as raw materials. This carbon dot material not only retains the high affinity of the fluorescent brightener group for fungi but also changes the physical and chemical properties of the fluorescent brightener. The prepared staining solution has the ability to remain stable in an aqueous solution for a long time, and the optical properties of fluorescein also change. The maximum excitation wavelength of the fluorescein carbon dots is 470 nm, and the maximum emission wavelength is 555 nm. Therefore, it can be observed under the blue light of 455 nm in a fluorescence microscope, causing the sample to be tested to exhibit a bright yellow fluorescence under blue light excitation. However, this carbon dot also has a relatively large emission in the blue light band. Therefore, when observed under the ultraviolet light of 365 nm in a fluorescence microscope, the sample to be tested can exhibit a cyan fluorescence under ultraviolet excitation, which is different from the conventional blue fluorescence of fluorescent brightener 135#; and the fluorescein carbon dot staining reagent has a simple composition, can quickly stain epidermal fungi, and can maintain a low background brightness of the sample tissue without combining with other complex reagents;

[0032] (5) Through the upgrade of the formula, the present invention adds an oxidant, ammonium persulfate, to achieve a strong affinity for fungi, enabling epidermal fungi to exhibit a tight spherical shape after staining, with a specific morphological structure different from the halo-like shape of dandruff. Therefore, it is possible to directly judge the infection situation of epidermal fungi without eliminating dandruff; using the fluorescein carbon dot staining solution in the present invention for fungal staining, after the fluorescein carbon dot material is mixed with a sample containing dandruff, etc., the hidden fungal cells can be marked with fluorescence and imaged under a fluorescence microscope. This method is simple and easy to implement, takes a short time, the obtained carbon dot fluorescence is stable, the fluorescence intensity is high, and the imaging effect on epidermal fungi is good; based on this, the prepared fluorescein carbon dot material in the present invention has the potential to develop into a general fluorescent dye for labeling epidermal fungi; therefore, the present invention provides a yellow-emitting fluorescein carbon dot staining reagent that can accurately judge the presence of epidermal fungi without eliminating dandruff, has a simple composition, can remain stable for a long time, and greatly reduces the cost. Description of the Drawings

[0033] The following further describes the present invention with reference to the drawings in conjunction with embodiments.

[0034] Figure 1 It is the fluorescence spectrum of the fluorescein carbon dots in Example 1. The maximum excitation wavelength is 470 nm, which can exhibit a cyan fluorescence under ultraviolet excitation and a bright yellow fluorescence under blue light excitation, different from the conventional fluorescence of fluorescent brightener 135#;

[0035] Figure 2It is the dual-color imaging of Saccharomyces cerevisiae after staining with fluorescein carbon dots in Example 1: In the 365 nm ultraviolet channel of the fluorescence microscope, the yeast appears cyan, and in the 455 nm blue channel, it appears yellow;

[0036] Figure 3 It is the dual-color imaging of untreated dandruff stained with fluorescein carbon dots in Example 2: The carbon dots were directly dropped onto the dandruff for imaging and then imaged under the fluorescence microscope. In the 365 nm ultraviolet channel of the fluorescence microscope, the yeast appears cyan, and in the 455 nm blue channel, it appears yellow;

[0037] Figure 4 It is the stability of fluorescein carbon dots in Example 3: After one month, the fluorescence brightness did not decrease significantly. Detailed implementation mode

[0038] The technical solutions of the present invention will be described in detail below in combination with specific embodiments, so that those skilled in the art can better understand and implement the technical solutions of the present invention. The following embodiments and drawings are only for illustrative purposes and should not be construed as limiting the present invention. The reagents or materials used in the embodiments are all from commercial channels unless otherwise specified. Unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

[0039] To describe the present invention in more detail, the following is expanded through embodiments. It should be emphasized that the following embodiments are only for explaining the present invention and are not used to limit the essential scope or content of the present invention.

[0040] Example 1

[0041] The fluorescein carbon dot staining solution for non-washing dual-color cell imaging for fungal detection provided in this example includes, by mass percentage: 1.00% of fluorescein carbon dot material and 99.00% of water.

[0042] The fluorescein carbon dot material is made from raw materials with the following mass percentages: 0.9% of fluorescent brightener 135#, 27% of ethylenediamine, 0.09% of ammonium persulfate, and 72.01% of water.

[0043] Among them:

[0044] The molecular formula of fluorescent brightener 135# is:

[0045]

[0046] The preparation method of the fluorescein carbon dot material is as follows:

[0047] According to the dosage relationship, 0.9% of fluorescent brightener 135#, 27% of ethylenediamine, 0.09% of ammonium persulfate, and 72.01% of water were selected, mixed evenly in a centrifuge tube, and then poured into a reaction kettle lined with polytetrafluoroethylene. Hydrothermal reaction was carried out in an oven at 140 °C for 10 h.

[0048] After the reaction, the liquid filtered through a 0.22 μm aqueous filter membrane is the obtained fluorescein carbon dot material.

[0049] The fluorescence spectrum of the aqueous solution of this carbon dot material is as Figure 1 shown. The fluorescence spectrum of fluorescein carbon dots: The maximum excitation wavelength is 470 nm. It can exhibit cyan fluorescence under ultraviolet excitation and bright fluorescence under blue light excitation, different from the conventional fluorescence of fluorescent brightener 135#, proving that the obtained fluorescein carbon dot material has the emission ability in the green to yellow light band.

[0050] The method for staining fungi using this fluorescein carbon dot staining reagent includes the following steps:

[0051] 1) Slide preparation: Take a clean glass slide, drop the Saccharomyces cerevisiae liquid to be tested in the middle area of the glass slide, and air-dry it to complete the slide preparation;

[0052] 2) Staining: Drop one drop of the fluorescent staining solution onto the glass slide for covering and stain for 10 min. The incubation temperature is the natural temperature, and it can be air-dried naturally or by heating;

[0053] 3) Sealing: Take a cover glass and directly cover the stained glass slide;

[0054] 4) Microscopic examination: Take the sample glass slide and observe it under the UV band and blue light band of a fluorescence microscope respectively to check the morphology and fluorescence intensity of the microorganisms in the microbial sample.

[0055] The result diagram of detecting Saccharomyces cerevisiae with fluorescein carbon dots is as Figure 2 shown. From Figure 2 it can be seen that the yeast fungi are successfully stained, with bright fluorescence, forming an obvious contrast with the dark background, which is conducive to the observation and identification of fungi, proving the fungal detection ability of fluorescein carbon dots.

[0056] Moreover, for the dual-color imaging of Saccharomyces cerevisiae after staining with fluorescein carbon dots, in the ultraviolet channel of the fluorescence microscope, the yeast appears cyan, and in the blue channel, it appears yellow.

[0057] Example 2

[0058] The fluorescein carbon dot staining solution for fungi detection and non-washing dual-color cell imaging provided in this example, calculated by mass percentage, includes: 1.00% of fluorescein carbon dot material and 99.00% of water.

[0059] The fluorescein carbon dot material is made from the following raw materials by mass percentage: fluorescent brightener 135# 0.9%, ethylenediamine 27%, ammonium persulfate 0.09%, and water 72.01%.

[0060] A method for directly detecting dandruff fungi of volunteers by using the fluorescein carbon dot staining reagent includes the following steps:

[0061] 1) Slide preparation: Take a clean glass slide, drop water in the middle area, move the dandruff of the volunteer to the water droplet, and dry it by heating with an alcohol lamp. The slide preparation is completed;

[0062] 2) Staining: Drop the fluorescein carbon dot staining reagent onto the sample to be tested for covering and staining for 10 min. The incubation temperature is the natural temperature, and it is air-dried naturally or by heating;

[0063] 3) Sealing: Take a cover glass and directly cover the stained glass slide;

[0064] 4) Microscopic examination: Take the sample slide and observe it under a fluorescence microscope in the UV and blue light bands to check the morphology and fluorescence intensity of the fungi in the sample to be tested.

[0065] The effect diagram of fluorescein carbon dots detecting epidermal fungi is as Figure 3 shown. It can be seen from Figure 3 that the epidermal fungi are successfully stained, showing a clear spherical shape, and showing a morphological structure different from that of dandruff. Dandruff is in the form of aggregates of a dozen or even hundreds of cells of different sizes. The fluorescein carbon dots have weak targeting ability to cells, so they show a smudged morphology under a fluorescence microscope, which is conducive to judging the scalp health status.

[0066] Moreover, the fluorescein carbon dot staining solution can perform dual-color imaging on untreated dandruff. The carbon dots are directly dropped onto the dandruff, without washing, and imaged under a fluorescence microscope. In the ultraviolet channel of the fluorescence microscope, yeast shows cyan, and in the blue channel, it shows yellow.

[0067] Example 3

[0068] The fluorescein carbon dot staining reagent for epidermal fungi detection provided in this example, by mass percentage, includes: 1.00% of fluorescein carbon dot material and 99.00% of water.

[0069] The fluorescein carbon dot material is made from the following raw materials by mass percentage: fluorescent brightener 135 0.9%, ethylenediamine 27%, ammonium persulfate 0.09%, and water 72.01%.

[0070] The fluorescence spectrum of the aqueous solution of this carbon dot material after one month of preparation shows that the fluorescence intensity has not changed significantly within one month. As Figure 4 shown, it proves that the material has high stability.

[0071] Example 4

[0072] Different from Example 1, the fluorescein carbon dot staining solution for non-washing two-color cell imaging for fungal detection provided in this example comprises, by mass percentage: 0.7% of fluorescein carbon dot material and 99.3% of water.

[0073] The fluorescein carbon dot material is made from raw materials with the following mass percentages: 0.5% of fluorescent brightener 135#, 25% of ethylenediamine, 0.07% of ammonium persulfate, and 74.43% of water.

[0074] Example 5

[0075] Different from Example 1, the fluorescein carbon dot staining solution for non-washing two-color cell imaging for fungal detection provided in this example comprises, by mass percentage: 1.50% of fluorescein carbon dot material and 98.50% of water.

[0076] The fluorescein carbon dot material is made from raw materials with the following mass percentages: 1% of fluorescent brightener 135#, 25% of ethylenediamine, 0.12% of ammonium persulfate, and 73.88% of water.

[0077] The above examples are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above examples. Those of ordinary skill in the art can achieve the purpose of the present invention based on the content disclosed above. Any improvements and modifications made on the basis of the concept of the present invention fall within the protection scope of the present invention, and the specific protection scope shall be subject to that recorded in the claims.

Claims

1. A non-washing two-color cell imaging fluorescein carbon dot staining solution for fungal detection, characterized in that: By mass percentage, it includes: 0.50% - 1.50% of fluorescein carbon dot material and 98.50% - 99.50% of water; wherein the fluorescein carbon dot material is made from raw materials with the following mass percentages: 0.5% - 1.3% of fluorescent brightener 135#, 25% - 29% of ethylenediamine, 0.05% - 0.13% of ammonium persulfate, and 69.57% - 74.45% of water.

2. The non-washing two-color cell imaging fluorescein carbon dot staining solution for fungal detection according to claim 1, wherein: By mass percentage, it includes: 1.00% of fluorescein carbon dot material and 99.00% of water; wherein the fluorescein carbon dot material is made from raw materials with the following mass percentages: 0.9% of fluorescent brightener 135#, 27% of ethylenediamine, 0.09% of ammonium persulfate, and 72.01% of water.

3. The non-washing two-color cell imaging fluorescein carbon dot staining solution for fungal detection according to claim 1, wherein: The fluorescein carbon dot material is prepared by a one-step hydrothermal reaction, including carrying out a hydrothermal reaction at a temperature of 120 - 180 °C for 8 - 12 hours.

4. The non-washing two-color cell imaging fluorescein carbon dot staining solution for fungal detection according to claim 3, wherein: The fluorescein carbon dot material is prepared by a one-step hydrothermal reaction, including carrying out a hydrothermal reaction in an oven at a temperature of 140 °C for 10 hours.

5. A method for fungal staining using the fluorescein carbon dot staining solution according to any one of claims 1-4, characterized in that It includes the following steps: 1) Slide preparation: Take a clean glass slide, drop water in the middle area, move the sample to be tested to the water drop, heat and dry it, and the slide preparation is completed. 2) Staining: Drop the fluorescein carbon dot staining solution onto the sample to be tested for covering and staining for 2 min - 10 min, and air dry. 3) Sealing the slide: Take a coverslip and directly cover the stained glass slide. 4) Microscopic examination: Take the sample slide and observe it under a fluorescence microscope in the UV light and blue light bands to check the morphology and fluorescence intensity of fungi in the sample to be tested.

6. Use of the fluorescein carbon dot staining solution according to any one of claims 1 - 4 in the detection of fungi by fluorescence staining method.

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