Anti-quenching fluorescent coloring agent as well as preparation method and application thereof

Abstract

The invention discloses an anti-quenching fluorescent coloring agent as well as a preparation method and application thereof. The anti-quenching fluorescent staining agent at least comprises concanavalin A, wheat germ agglutinin, quantum dots and a bacteriostatic agent. The anti-quenching fluorescent staining agent disclosed by the invention ensures that stained fungi are in an active infection stage and have strong pathogenicity by utilizing the characteristics of lectin; the quantum dot is used as a chromogenic group, the selected wavelength is wider, the fluorescence quenching is low, the chromogenic time is long, the clinical diagnosis time is ensured, the problems of missing detection and difficult detection caused by short fluorescence duration in clinic are solved, and an efficient, stable and accurate method is provided for clinical live fungal infection diagnosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to an anti-fading fluorescent dye and a preparation method and application thereof. Background Art

[0002] Fungi are a class of microorganisms that include yeasts and molds. Fungal infections usually occur on the skin, nails, hair, and mucous membranes. Common fungal infections include Candida albicans infections (oral Candida infections, vaginal Candida infections), tinea pedis, and nail fungal infections. Some fungal infections may require drug treatment, and severe infections may require systemic antifungal drugs.

[0003] In the diagnosis and treatment of fungal infections, accurate and rapid detection of active fungi is crucial. Some common fungal detection techniques include culture and separation techniques, molecular biology methods, fluorescent staining, mass spectrometry, immunological methods, and rapid detection techniques.

[0004] Fluorescence detection is a rapid fungal detection method that has been widely used. Traditional staining methods have problems such as time-consuming staining process, unstable staining effect, high missed detection rate, poor dye stability, and fluorescence quenching. Therefore, it is necessary to develop a new method to improve the accuracy and stability of fungal detection.

[0005] Quantum dot labeling technology is an advanced imaging technology used in the biomedical field. It uses nanoscale semiconductor material quantum dots as fluorescent markers to label biological molecules or cell structures to achieve high-sensitivity and high-resolution imaging. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide an antifade fluorescent stain and its preparation method and application. The antifade fluorescent stain of the present invention can be used for the detection of live fungi, and can realize quench-free fluorescence microscopy staining observation of pathogenic fungi. The present invention combines the specificity of concanavalin A and wheat germ agglutinin with the stability of quantum dots to ensure accurate judgment of the active stage of fungal infection. The present invention provides an efficient, stable and accurate method for the detection of live fungi, can be widely used in fungal research, and has potential clinical application prospects.

[0007] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0008] The present invention provides an anti-fading fluorescent stain, wherein the stain at least comprises concanavalin A, wheat germ agglutinin, quantum dots and an anti-fading agent;

[0009] The proportion of concanavalin A in the dye is 0.1-1 mg / 100 mL;

[0010] The proportion of wheat germ agglutinin in the dye is 0.1-1 mg / 100 mL;

[0011] The quantum dots account for 0.1-0.5 mg / 100 mL of the dye;

[0012] The anti-quenching agent accounts for 80-180 mg / 100 mL in the staining agent.

[0013] The concanavalin A binds to A-mannopyranosyl and A-glucopyranosyl residues in the fungal cell wall. The wheat germ agglutinin has a high affinity for sialic acid and N-acetylglucosamine and can bind to budding fungi.

[0014] The concanavalin A of the present invention can specifically bind to glycosyl residues in yeast and fungal cell walls, and wheat germ agglutinin can stain active budding fungi. By labeling concanavalin A and wheat germ agglutinin with quantum dots, the detection of active fungi can be achieved.

[0015] Semiconductor quantum dot materials are usually binary or ternary semiconductor compounds of II-VI or III-V groups. The quantum dots of the present invention can be selected from common quantum dot materials, such as ZnTe, CdSe, CdS, CdTe, etc., and can also be selected from quantum dots with shells or modified, such as CdSe / ZnS quantum dots, CdSe / ZnS / SiO 2 Quantum dots, etc., select the luminescent color and excitation wavelength.

[0016] The present invention applies quantum dot materials to clinical infectious fungus detection. Compared with existing fluorescent staining, Gram staining and "white film", it has the characteristics of no quenching and easy observation and diagnosis.

[0017] Preferably, the quantum dots are selected from ZnTe quantum dots, CdSe quantum dots, thioglycolic acid-modified ZnTe quantum dots or thioglycolic acid-modified CdSe quantum dots.

[0018] Further preferably, the quantum dots are selected from thioglycolic acid-modified ZnTe quantum dots (excitation wavelength 365nm, emission wavelength 425nm) or thioglycolic acid-modified CdSe quantum dots (excitation wavelength 365nm, emission wavelength 620nm). ZnTe and CdSe quantum dots have excellent fluorescence quantum yield, narrow emission bandwidth, and excellent fluorescence performance; the excitation wavelength of 365nm is the most common and easy to obtain, which increases the applicability of the present invention. The quantum dots selected by the present invention have a long luminescence duration and no fluorescence quenching phenomenon.

[0019] Preferably, the anti-quenching agent is one or more of P-phenylene diamine (PPD), n-propyl gallate (NPG), and 1,4-diazobicyclo[2,2,2]-octane (1,4diazobicyelo(2,2,2)-octane, DABCO).

[0020] A preferred embodiment of the present invention is that the dye may further include one or more of an antibacterial agent, an organic cosolvent, a moisturizer or a stabilizer. By utilizing the anti-fading property of quantum dots and adding an anti-fading protective agent and a background eliminator, a long-term stable test of active fungal infection can be achieved.

[0021] Furthermore, the antibacterial agent is one or more of thimerosal, sodium azide or ProClin 300. Thimerosal has strong antibacterial ability against both Gram-positive and Gram-negative bacteria and is a broad-spectrum antibacterial agent. Sodium azide is one of the most commonly used preservatives in in vitro diagnostic reagents. ProClin 300 activity has a broad spectrum of applicability. The proportion of the antibacterial agent in the dye is 0.05-1g / 100mL.

[0022] Furthermore, the organic cosolvent is glycerol, which accounts for 2-10mL / 100mL in the stain. Glycerol is a polar organic solvent that can dissolve and disperse the target compound well, thereby improving its solubility and bioavailability in aqueous solution. This is very important for the application of fungal antifading fluorescent stains. At the same time, glycerol is a relatively safe and non-toxic organic compound that is widely used in biological applications. Compared with some other organic solvents, glycerol has less effect on biological systems, which is beneficial to improving the biocompatibility of the stain. Glycerol has good chemical stability and can protect the target compound from damage by environmental factors such as oxidation and hydrolysis, thereby improving the stability and service life of the stain. In addition, glycerol can also play the role of a moisturizer.

[0023] Wetting agent of the present invention can be selected from dimethyl sulfoxide, glycerine, propylene glycol, sorbitol or polyethylene glycol (PEG). Wherein, glycerine, as a kind of polyhydric alcohol, has excellent moisture retention performance, can absorb and keep moisture, improves the wettability and stability of coloring agent. Propylene glycol is also a kind of common wetting agent, has good solubility and permeability, can enhance the wettability and permeability of coloring agent. Sorbitol is a kind of polyol wetting agent, has strong hygroscopicity and moisture retention ability, helps to maintain the moist state of coloring agent. PEG of different molecular weight can be used as wetting agent, has good hydrophilicity and moisture retention.

[0024] Further, the wetting agent is dimethyl sulfoxide (DMSO). Dimethyl sulfoxide is a colorless liquid, odorless or almost odorless, hygroscopic, and is called a universal solvent. DMSO has certain hygroscopicity and can absorb water molecules from the environment to increase its own water content. The DMSO molecule is small and has good permeability, and can penetrate into the skin or other surfaces to carry water molecules. DMSO can form a protective film on the skin or surface to block the evaporation of water, thereby maintaining the wet state of the surface. The DMSO molecule has certain affinity and can form hydrogen bonds with water molecules to enhance the retention capacity of water molecules. Therefore, DMSO can be used as an effective wetting agent, and plays a role in keeping the surface moist and preventing water evaporation. The proportion of the wetting agent in the dye is 10-20mL / 100mL.

[0025] The stabilizer of the present invention can be selected from sorbitol, other sugar stabilizers (such as sucrose and glycerol), and surfactants (such as Tween-20, SDS).

[0026] Furthermore, the stabilizer is sorbitol. Sorbitol is a common carbohydrate stabilizer, which is widely used in the field of biotechnology and has mature experience in use. Sorbitol has good solubility and compatibility and will not interfere with sensitive fluorescent dyes or enzymes. Sorbitol can increase the viscosity and moisture retention of the solution and reduce the evaporation and degradation of the reagent. Sorbitol works synergistically with other ingredients to improve the stability and shelf life of the overall formula. The proportion of the stabilizer in the dye is 3-20 mg / 100 mL.

[0027] The present invention also provides a method for preparing the anti-fading fluorescent dye, comprising the following steps:

[0028] (1) Weigh concanavalin A and wheat germ agglutinin, add them to water, place at 4°C and mix thoroughly, and adjust the solution pH to 8.0±0.2;

[0029] (2) Weighing quantum dots and adding them to the solution obtained in step (1), stirring thoroughly, and leaving at 4°C to react overnight;

[0030] (3) Weigh an antifade agent, an antibacterial agent, an organic solubilizing agent, a humectant, and a stabilizer and add them to the solution obtained in step (2), add water, and adjust the volume to obtain the antifade fluorescent dye, which is stored in a dark place at 4°C.

[0031] Preferably, in step (1), 0.1 M sodium hydroxide is used to adjust the solution to pH = 8.0 ± 0.2.

[0032] The present invention also provides application of the anti-fading fluorescent dye in live fungus detection.

[0033] The detection method using the anti-fading fluorescent dye comprises the following steps:

[0034] (1) Place the test sample in the middle observation area of ​​the slide;

[0035] (2) Add an appropriate amount of the antifade fluorescent dye to completely immerse the sample, cover it with a coverslip, and observe it using an ultraviolet flashlight or 365 nm excitation light under a fluorescence microscope.

[0036] The detection method of the present invention can be observed entirely using the UV band.

[0037] Beneficial effects:

[0038] 1. The present invention adds concanavalin A and wheat germ agglutinin, which can specifically bind to the surface of fungi, eliminate bacteria and abnormal cells in time, and improve the accuracy of detection.

[0039] 2. The present invention adds quantum dot labels. Compared with traditional organic fluorescent substances, quantum dots only need a single excitation wavelength to achieve one or more band detection, and there is no fluorescence quenching, the duration is long, and the stability of detection is improved.

[0040] 3. The present invention adopts a one-step staining method, which is easy to operate and has a short operation time. The test results can be observed quickly without delaying the patient's condition and treatment medication.

[0041] 4. Compared with traditional fungal diagnosis and testing methods, the anti-fading fluorescent stain of the present invention has the following advantages:

[0042] (1) By utilizing the characteristics of lectins, the stained fungi are ensured to be in the active infection stage, and highly pathogenic fungal samples can be screened;

[0043] (2) Using quantum dots as the color-developing group, the observed wavelength is wider, the fluorescence quenching is low, and the color development time is long, which ensures the time of clinical diagnosis and solves the problems of missed detection and difficulty in detection caused by the short fluorescence duration in clinical practice, providing an efficient, stable and accurate method for the diagnosis of clinical live fungal infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a microscopic observation effect diagram of Candida albicans staining with the anti-fading fluorescent stain prepared in Example 1 of the present invention for 15 minutes.

[0045] Figure 2 This is a microscopic observation effect diagram of Candida albicans staining with the anti-fading fluorescent stain prepared in Example 2 of the present invention for 15 minutes.

[0046] Figure 3 This is a microscopic observation effect diagram of Candida albicans staining with the anti-fading fluorescent stain prepared in Example 3 of the present invention for 15 minutes.

[0047] Figure 4 This is the effect of Candida albicans Gram staining under microscope.

[0048] Figure 5 This is the effect of Candida albicans KOH white film method under microscope observation.

[0049] Figure 6 The staining results of Candida albicans using the common fluorescent method (a is the staining result at 0 minutes, b is the staining result at 5 minutes, and c is the staining result at 15 minutes).

[0050] Figure 7 The Candida albicans staining results of the anti-fading fluorescent stain prepared in Example 1 of the present invention (a is the staining result at 0 minutes, and b is the staining result at 15 minutes). DETAILED DESCRIPTION

[0051] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0052] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0053] Example 1 Preparation of antifade fluorescent dye

[0054] Thioglycolic acid-modified ZnTe quantum dots were purchased from Qiyue Biotechnology with an excitation wavelength of 365 nm and an emission wavelength of 425 nm.

[0055] Weigh concanavalin A and wheat germ agglutinin into a beaker, add 10 mL of ultrapure water, place at 4 ° C to mix thoroughly, and use 0.1 M sodium hydroxide to adjust the solution to pH = 8.0; weigh thioglycolic acid-modified ZnTe quantum dots and add them to the solution, stir thoroughly, and place at 4 ° C to react overnight; add glycerol, sorbitol, dimethyl sulfoxide, n-propyl gallate and ProClin 300 to the overnight reaction solution, use a volumetric flask, and dilute to 100 mL with ultrapure water to obtain the anti-fading fluorescent dye, which is stored in a dark place at 4 ° C.

[0056] The antifade fluorescent dye contains: 0.1 mg of concanavalin A, 0.1 mg of wheat germ agglutinin, 0.1 mg of quantum dots, 2 mL of glycerol, 3 mg of sorbitol, 10 mL of dimethyl sulfoxide, 80 mg of n-propyl gallate, 0.05 g of ProClin 3000, and the balance is ultrapure water.

[0057] Candida albicans (ATCC 10231) cultured overnight (YPD medium, 30°C) was diluted with PBS to an OD600 of 0.2, 5 μl was pipetted onto a glass slide and evenly coated, a drop of the above fluorescent dye was added, and a coverslip was placed on the slide. The blue light quenching was observed under a fluorescence microscope using the UV band for 15 minutes (see Figure 1 ),from Figure 1 It can be seen that there is basically no quenching of fluorescence and obvious budding can be observed.

[0058] Example 2 Preparation of antifade fluorescent stain

[0059] Thioglycolic acid-modified CdSe quantum dots were purchased from Qiyue Biotechnology with an excitation wavelength of 365 nm and an emission wavelength of 620 nm.

[0060] Weigh concanavalin A and wheat germ agglutinin into a beaker, add 10 mL of ultrapure water, place at 4 ° C to mix thoroughly, and use 0.1 M sodium hydroxide to adjust the solution to pH = 7.8; weigh thioglycolic acid-modified CdSe quantum dots and add them to the solution, stir thoroughly, and place at 4 ° C to react overnight; add glycerol, sorbitol, dimethyl sulfoxide, n-propyl gallate and ProClin 300 to the overnight reaction solution, use a volumetric flask, and dilute to 100 mL with ultrapure water to obtain the anti-fading fluorescent dye, which is stored at 4 ° C away from light.

[0061] The antifade fluorescent stain contains: 1 mg of concanavalin A, 1 mg of wheat germ agglutination, 0.5 mg of quantum dots, 10 mL of glycerol, 20 mg of sorbitol, 20 mL of dimethyl sulfoxide, 180 mg of n-propyl gallate, 1 g of ProClin 300, and the balance is ultrapure water.

[0062] The Candida albicans cultured overnight (YPD medium, 30°C) was diluted with PBS to an OD600 of 0.1, 5 μl was pipetted onto a glass slide and evenly coated, a drop of the above fluorescent dye was added, and a coverslip was covered. The red light quenching was observed under a fluorescence microscope using the UV band for 15 minutes (see Figure 2 ),from Figure 2 It can be seen that there is basically no quenching of fluorescence and obvious budding can be observed.

[0063] Example 3 Preparation of anti-fading fluorescent dye

[0064] Thioglycolic acid-modified ZnTe quantum dots were purchased from Qiyue Biotechnology with an excitation wavelength of 365 nm and an emission wavelength of 425 nm.

[0065] Weigh concanavalin A and wheat germ agglutinin into a beaker, add 10 mL of ultrapure water, place at 4 ° C to mix thoroughly, and use 0.1 M sodium hydroxide to adjust the solution to pH = 8.2; weigh thioglycolic acid-modified ZnTe quantum dots and add them to the solution, stir thoroughly, and place at 4 ° C to react overnight; add glycerol, sorbitol, dimethyl sulfoxide, n-propyl gallate and ProClin 300 to the overnight reaction solution, use a volumetric flask, and dilute to 100 mL with ultrapure water to obtain the anti-fading fluorescent dye, which is stored at 4 ° C away from light.

[0066] The antifade fluorescent stain contains: 0.55 mg of concanavalin A, 0.55 mg of wheat germ agglutinin, 0.3 mg of quantum dots, 6 mL of glycerol, 11.5 mg of sorbitol, 15 mL of dimethyl sulfoxide, 130 mg of n-propyl gallate, 0.525 g of ProClin 300, and the balance is ultrapure water.

[0067] Candida albicans (ATCC 10231) cultured overnight (YPD medium, 30°C) was diluted with PBS to an OD600 of 0.15, 5 μl was pipetted onto a glass slide and evenly spread, a drop of the above fluorescent dye was added, and a coverslip was placed on the slide. The blue light quenching was observed under a fluorescence microscope using the UV band for 15 minutes (see Figure 3 ), it can be seen from the figure: there is basically no fluorescence quenching, and obvious budding can be observed.

[0068] Example 4 Staining of Candida albicans

[0069] Candida albicans cultured overnight (YPD medium, 30°C) was diluted to OD600 of 0.3, and 5 μl of each sample was evenly applied to a glass slide to prepare 4 specimens. The specimens were stained with the antifade fluorescent stain prepared in Example 1, fungal fluorescent stain (Sankai Medical), Gram stain, and KOH wet mount microscopy for observation.

[0070] Generally, Gram staining can be used to classify bacteria into two categories: Gram-positive bacteria and Gram-negative bacteria. Gram staining can also be used for fungal detection. When Gram staining is used, fungi are stained positive, such as Candida, yeast, and spore-tricholoma.

[0071] The classic Gram stain reagent consists of the following:

[0072] 1) Primary staining: Crystal violet staining solution (composed of crystal violet and ammonium oxalate solution)

[0073] 2) Mordant: Gram iodine solution (composed of iodine and potassium iodide solution)

[0074] 3) Decolorization: 95% ethanol or 3:7 acetone ethanol

[0075] 4) Re-staining: Sand yellow (composed of sand yellow, ethanol and water)

[0076] The steps for Gram staining are as follows:

[0077] Primary dyeing → washing → coal dyeing → washing → bleaching → washing → re-dyeing → washing → drying.

[0078] Gram staining and wet mount microscopy were directly observed under an ordinary microscope, and the Candida staining results could be seen. The anti-fading fluorescent staining and fungal fluorescent staining prepared in Example 1 of the present invention were observed in the UV band under a fluorescent microscope, and the quenching was observed for 15 minutes. The results showed that the Candida observed in the four specimens had similar morphologies, which were round or oval, but the staining effects were different.

[0079] Gram staining stains Candida into purple, but it is not specific and the procedure is cumbersome. Figure 4 Wet mount microscopy is relatively simple to operate, but difficult to observe. The staining results are shown in Figure 5 The fungal fluorescent staining method and the anti-fading fluorescent staining agent prepared in Example 1 of the present invention can both make Candida appear blue, but the fluorescence of ordinary fluorescent staining is basically quenched after 5 minutes. The staining results are as follows Figure 6 As shown. However, when the anti-fading fluorescent dye prepared in Example 1 of the present invention was used for staining for 15 minutes, the fluorescence was basically not quenched, and obvious budding could be observed. The staining results are shown in Figure 7 This indicates that the anti-fading fluorescent dye prepared by the present invention has a long duration for detecting living fungi and improves the stability of detection.

[0080] It can be seen that compared with the fungal detection methods currently used in clinical practice, the present invention solves the shortcomings of clinical fungal detection:

[0081] 1. Traditional white slides (usually refers to microscope slides that have not been stained or treated and are used to directly observe samples) are difficult to observe, while the direct fluorescent staining of the present invention is easy to observe.

[0082] 2. Gram staining is a complicated operation, while the fluorescent staining of the present invention is done in one step.

[0083] 3. Fluorescein staining quenches quickly (within 5 minutes), and the observation time is short, which makes it difficult to ensure clinical diagnosis observation. The present invention introduces quantum dots and anti-quenching agents to achieve long-term observation after staining.

[0084] 4. Fluorescence staining has no specificity. The present invention introduces concanavalin A to ensure specific binding with fungal cell wall components.

[0085] 5. Infectious fungi are pathogenic only when they germinate. The present invention can ensure that the fungi are in the infection stage and are pathogenic by using wheat germ agglutinin, thus avoiding misdiagnosis.

[0086] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. An anti-fading fluorescent dye, characterized in that: The dye at least comprises concanavalin A, wheat germ agglutinin, quantum dots and an antifade agent; The proportion of concanavalin A in the dye is 0.1-1 mg / 100 mL; The proportion of wheat germ agglutinin in the dye is 0.1-1 mg / 100 mL; The quantum dots account for 0.1-0.5 mg / 100 mL of the dye; The anti-quenching agent accounts for 80-180 mg / 100 mL in the staining agent.

2. The anti-fading fluorescent dye according to claim 1, characterized in that The quantum dots are selected from ZnTe quantum dots, CdSe quantum dots, ZnTe quantum dots modified with thioglycolic acid or CdSe quantum dots modified with thioglycolic acid.

3. The anti-fading fluorescent dye according to claim 1, characterized in that The anti-quenching agent is one or more of P-phenylenediamine PPD, n-propyl gallate NPG, and 1,4-diazobicyclo[2,2,2)-octane DABCO.

4. The anti-fading fluorescent dye according to claim 1, characterized in that The dyeing agent further comprises one or more of an antibacterial agent, an organic solubilizing agent, a moisturizing agent or a stabilizer.

5. The anti-fading fluorescent dye according to claim 4, characterized in that: The antibacterial agent is one or more of thimerosal, sodium azide or proclin 300; The antibacterial agent accounts for 0.05-1g / 100mL in the dye.

6. The anti-fading fluorescent dye according to claim 4, characterized in that: The organic dissolving agent is glycerol, and its proportion in the dye is 2-10mL / 100mL.

7. The anti-fading fluorescent dye according to claim 4, characterized in that: The moisturizing agent is dimethyl sulfoxide, and the stabilizer is sorbitol; the proportion of the moisturizing agent in the dye is 10-20 mL / 100 mL; and the proportion of the stabilizer in the dye is 3-20 mg / 100 mL.

8. A method for preparing the anti-fading fluorescent dye according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Weigh concanavalin A and wheat germ agglutinin, add them to water, place at 4°C and mix thoroughly, and adjust the solution pH to 8.0±0.2; (2) Weighing quantum dots and adding them to the solution obtained in step (1), stirring thoroughly, and leaving at 4°C to react overnight; (3) Weigh an antifade agent, an antibacterial agent, an organic solubilizing agent, a humectant, and a stabilizer and add them to the solution obtained in step (2), add water, and adjust the volume to obtain the antifade fluorescent dye, which is stored in a dark place at 4°C.

9. Use of the antifade fluorescent dye according to any one of claims 1 to 7 in the detection of living fungi.

10. The use according to claim 9, characterized in that: The detection method using the anti-fading fluorescent dye comprises the following steps: (1) Place the test sample in the middle observation area of ​​the slide; (2) Add the antifade fluorescent dye dropwise until the sample is completely immersed, cover with a coverslip, and observe using an ultraviolet flashlight or 365 nm excitation light under a fluorescence microscope.

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