Preparation method of fungal fluorescent probe and obtained product

Abstract

The invention discloses a preparation method and obtained products of a fungal fluorescent probe, wherein chloroauric acid and sodium chloride are dissolved in water, then ventilated and left to stand until crystals are completely precipitated to obtain sodium chloride crystals containing chloroauric acid; crystals and a graphite phase carbon nitride precursor are ground and pre-treated, and then the temperature is increased for a first heat treatment to obtain layered g-C3N4 nanosheets embedded with gold nanoparticles; layered g-C3N4 nanosheets embedded with gold nanoparticles are ground and pre-treated with an organic acid containing a sulfhydryl group, and then the temperature is increased for a second heat treatment, the obtained sample is washed, then ultrasonically dispersed in water, and a suspension is centrifuged after ultrasound to obtain a fungal fluorescent probe. The method of the present invention is simple in process and highly specific, can be used for the diagnosis of various fungi, has a good application prospect in the field of early diagnosis of various skin diseases, and can also link other specific recognition molecules for other detection fields.

Description

Technical Field

[0001] The present invention relates to a preparation method of a fungal fluorescent probe and the obtained product, and specifically to a preparation method of a fungal fluorescent probe with high luminescence efficiency, good specificity and surface modified carboxyl groups and the obtained fungal fluorescent probe, belonging to the field of luminescent materials and application technology. Background Art

[0002] Fluorescent probes are a technique for observing the fluorescence of specific markers by utilizing the luminescence of materials after exposure to light. Their nature is closely related to the luminescent material. There are many types of fluorescent probes, including various luminescent organic molecules, semiconductor quantum dots, and carbon dots. Fluorescent probes are widely used in various detection and labeling applications, including measuring metal ions, pesticide residues, biomolecule content, tracing biomolecules, and labeling macromolecules, cellular, and subcellular structures. In recent years, they have also seen rapid development in the fields of medicine and disease control, finding widespread applications in areas such as fungal and microbial detection.

[0003] Fungal infection refers to a disease caused by one or more pathogenic fungi invading body tissues and resulting from the fungi or their metabolites. Due to limited understanding of the biological characteristics of pathogenic fungi and the diverse clinical manifestations of fungal infections, missed and misdiagnoses are common, leading to worsening of the condition and even life-threatening outcomes. Fungal morphological immunofluorescence diagnosis is a new and effective method for rapid and effective fungal detection. It not only provides a basis for accurate and rapid clinical diagnosis of fungal infections, but also facilitates rapid diagnosis and treatment, as well as rational medication use. It has significant social value in protecting human health and enhancing the body's immune system.

[0004] Fungal morphological immunofluorescence diagnosis is a new diagnostic technique for the rapid detection of fungi. Applying immunological and morphological principles, the β-D-glucan binding protein contained in the reagent specifically binds to glucans on the fungal cell wall. Through fluorescent labeling, the fungi are stained, emitting bright fluorescence under ultraviolet light. Their morphology can be observed using a fluorescence microscope, allowing for rapid detection of fungi. Carbon-based luminescent materials have important applications in biosensors, medical imaging devices, and light-emitting diodes due to their low toxicity and good biocompatibility. Carbon nitride is commonly used as a visible light photocatalytic material and is widely used in the green energy sector, such as photocatalytic water splitting to produce hydrogen, CO2 reduction, and nitrogen photoelectrocatalytic ammonia production. Compared with conventional luminescent carbon dots, carbon nitride materials have a simpler preparation process and higher stability. Therefore, the development of luminescent carbon nitride materials for use as fluorescent probes has become a focus of attention. However, carbon nitride has low biocompatibility and requires improvement before it can be used in fluorescent probes. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a fungal fluorescent probe and the resulting product. The preparation process of the method is controllable, reproducible, and convenient for industrial production. The resulting fungal fluorescent probe can emit a single green light with high luminescence brightness, has carboxyl groups on the surface, has good biocompatibility, and has strong application potential.

[0006] The specific technical solutions of the present invention are as follows:

[0007] A method for preparing a fungal fluorescent probe comprises the following steps:

[0008] (1) Dissolve chloroauric acid and sodium chloride in water, then ventilate and let stand until crystals are completely precipitated to obtain sodium chloride crystals containing chloroauric acid;

[0009] (2) mixing sodium chloride crystals containing chloroauric acid and a graphite-phase carbon nitride precursor and performing a grinding pretreatment;

[0010] (3) The mixture obtained after grinding is heated to perform a first heat treatment to obtain layered g-C3N4 nanosheets embedded with gold nanoparticles;

[0011] (4) The layered g-C3N4 nanosheets embedded with gold nanoparticles are mixed with an organic acid containing a thiol group, subjected to a grinding pretreatment, and then subjected to a second heat treatment by heating;

[0012] (5) The sample after the second heat treatment is washed and then ultrasonically dispersed in water. After ultrasonication, the suspension is centrifuged to obtain a fungal fluorescent probe.

[0013] Furthermore, in step (1), the mass ratio of chloroauric acid to sodium chloride is 0.01-0.02:1, for example, 0.010:1, 0.011:1, 0.012:1, 0.013:1, 0.014:1, 0.015:1, 0.016:1, 0.017:1, 0.018:1, 0.019:1, and 0.02:1. Chloroauric acid and sodium chloride are first dissolved in water to form a solution, which is then placed in a natural environment with ventilation for recrystallization. The purpose of recrystallization is to uniformly grow chloroauric acid in sodium chloride crystals. The amount of water used ensures that the sodium chloride concentration is near saturation, for example, it can be close to saturation, saturation, or slightly supersaturated. Preferably, the ratio of sodium chloride to water is 2g:5-6ml.

[0014] Furthermore, in step (2), the graphite phase carbon nitride precursor is melamine or dicyandiamide.

[0015] Furthermore, in step (2), the mass ratio of the graphite phase carbon nitride precursor to the sodium chloride crystals containing chloroauric acid in step (1) is 1-2:0.3-0.5, for example, 1:0.3, 1:0.4, 1:0.5, 2:0.3, 2:0.4, 2:0.5.

[0016] Furthermore, in step (2), the grinding time is 5-10 hours, for example, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, and 10 hours. The purpose of grinding is to ensure sufficient pre-reaction of the solid phase material through mechanical force.

[0017] Furthermore, in step (3), a first heat treatment undergoes a thermal polymerization reaction, which can directly embed the gold nanoparticles into the g-C3N4 nanosheets. The first heat treatment is performed under an inert atmosphere, which can be achieved by an inert gas such as nitrogen or argon. The first heat treatment temperature is 550-650 degrees Celsius, for example, 550°C, 600°C, or 650°C. The treatment time is generally 2-4 hours, for example, 2 hours, 3 hours, or 4 hours.

[0018] Furthermore, in step (4), the organic acid containing a thiol group is thioglycolic acid or thiopropionic acid.

[0019] Furthermore, in step (4), the mass ratio of the organic acid containing thiol groups to the layered g-C3N4 nanosheets embedded with gold nanoparticles (Au-g-C3N4 nanosheets) is (5-10)%:1, for example, 5%:1, 6%:1, 7%:1, 8%:1, 9%:1, and 10%:1.

[0020] Furthermore, in step (4), the grinding time is 4-8 hours, for example, 4 hours, 5 hours, 6 hours, 7 hours, and 8 hours. The purpose of grinding is also to complete the pre-reaction of the solid phase material.

[0021] Furthermore, in step (4), a second heat treatment is performed to undergo a thermal polymerization reaction, the purpose of which is to modify the thiol groups on the surface of the layered g-C3N4 nanosheets embedded with the gold nanoparticles, thereby forming binding sites for the fungus-specific β-D-glucan binding protein. The second heat treatment temperature is 300-400 degrees Celsius, for example, 300°C, 350°C, or 400°C. The treatment time is generally 1-2 hours. The second heat treatment is also performed under an inert atmosphere, which can be achieved by an inert gas such as nitrogen or argon.

[0022] Furthermore, in step (5), after heat treatment, the obtained sample is ground, then washed with water and alcohol, and then placed in water for ultrasonic treatment, with the mass ratio of sample to water being 20-50%:1, for example, 20%:1, 30%:1, 40%:1, or 50%:1. The purpose of ultrasonic treatment is to evenly disperse the sample in water to obtain a suspension, and ultrasonic treatment can be performed using commonly used laboratory ultrasonic equipment. After ultrasonic treatment, the suspension is centrifuged at a speed of 3000-4000 rpm, for example, 3000 rpm, 3100 rpm, 3200 rpm, 3300 rpm, 3400 rpm, 3500 rpm, 3600 rpm, 3700 rpm, 3800 rpm, 3900 rpm, or 4000 rpm, for about 10-20 minutes. After centrifugation, the suspension at the top is the suspension containing the fungal fluorescent probe.

[0023] The fungal fluorescent probe obtained according to the above method is a high-brightness, monochromatic green fluorescent probe with stable luminescence performance. Due to the surface modification of carboxyl groups, it can be covalently linked to β-D-glucan binding protein for fungal detection. It also has good application prospects in other biological probes, fluorescent tracing, luminescence and other fields.

[0024] The present invention prepares a solution of chloroauric acid and sodium chloride, followed by recrystallization to uniformly grow the chloroauric acid within sodium chloride crystals. The recrystallized crystals are then ground with graphite-phase carbon nitride for pretreatment. Small gold nanoparticles are then directly embedded within g-C3N4 nanosheets using a molten salt thermal polymerization method (550-650°C). The resulting material is then ground with a thiol-containing organic acid for pretreatment and then heat-treated (300-400°C) to produce a fungal fluorescent probe with surface-modified carboxyl groups. This fluorescent probe exhibits high luminescence brightness and excellent monochromaticity. The surface carboxyl groups can be covalently linked to a fungus-specific β-D-glucan binding protein, thereby enabling specific fluorescent detection of fungi. This method is simple and highly specific, making it suitable for the diagnosis of various fungi and has promising applications in the early diagnosis of various skin diseases. It can also be linked to other specific recognition molecules for other detection applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an electron microscope photograph of the sample obtained in Example 1, and the inset is an electron microscope photograph of a gold cluster.

[0026] Figure 2 This is the fluorescence spectrum of the sample obtained in Example 1. DETAILED DESCRIPTION

[0027] The present invention will be further described below by way of examples. It should be understood that the following description is only for the purpose of explaining the present invention and does not limit its contents.

[0028] Example 1

[0029] 1.1 Add 5 mL of water to a beaker, then dissolve 0.04 g of chloroauric acid and 2 g of NaCl in 5 mL of water (heat slightly until completely dissolved) and stir thoroughly. Cover the beaker with plastic wrap, leaving a small hole for water evaporation. Place in a fume hood. After 48 hours, remove the precipitated crystals, dry them, and set aside.

[0030] 1.2 Take 2 g of the crystals from step 1.1 and grind them with 5 g of melamine for 7 hours. Mix well and turn them into a fine powder for later use.

[0031] 1.3 Take 6 g of the fine powder from step 1.2 and treat it at 600°C under argon atmosphere for 3 hours. Then wash it with water 3 times and ethanol 3 times, and dry it for later use.

[0032] 1.4 Take 5 g of the sample from step 1.3, add 0.35 g of thioglycolic acid, and grind for 6 hours to mix evenly. After treating at 350°C under argon atmosphere for 1.5 hours, wash with water and ethanol three times each. Then, ultrasonically disperse the sample in water at a mass ratio of sample to water of 35%:1 to obtain a suspension.

[0033] 1.5 Take the suspension sample from step 1.4 and centrifuge it at 3500 rpm for 20 minutes. The suspension at the top is the prepared fungal fluorescent probe. Figure 1 This is an electron microscope image of the sample. The inset shows the gold lattice phase, indicating that the Au nanoparticles are embedded in the carbon nitride network. Figure 2 This is the fluorescence spectrum of the obtained fungal fluorescent probe. It can be seen from the figure that its emission spectrum is a single green, the luminescence is symmetrical, and the stability is good.

[0034] Example 2

[0035] 2.1 Add 5 mL of water to a beaker, then dissolve 0.02 g of chloroauric acid and 2 g of NaCl in 5 mL of water and stir thoroughly. Cover the beaker with plastic wrap, leaving a small hole for water evaporation. Place in a fume hood. After 48 hours, remove the precipitated crystals, dry them, and set aside.

[0036] 2.2 Grind 5g of melamine and 2g of the crystals obtained in step 2.1 for 10 hours, mix well and form a fine powder for later use;

[0037] 2.3 Take 6 g of the fine powder from step 2.2 and treat it at 550°C under nitrogen atmosphere for 4 hours. Then wash it with water 3 times and ethanol 3 times, and dry it for later use.

[0038] 2.4 Take 5 g of the sample from step 2.3, add 0.35 g of thioglycolic acid, and grind and mix thoroughly. After treating at 300 °C under a nitrogen atmosphere for 2 hours, wash with water and ethanol three times each. Then, ultrasonically disperse the sample in water at a mass ratio of 50%:1 to obtain a suspension.

[0039] 2.5 Take the suspension sample from step 2.4 and centrifuge it at 3500 rpm for 30 minutes. The upper suspension is the prepared fungal fluorescent probe. The luminescence color and microstructure of the resulting sample are similar to those in Example 1, and the sample exhibits bright green luminescence.

[0040] Example 3

[0041] 3.1 Add 5 mL of water to a beaker, then dissolve 0.03 g of chloroauric acid and 2 g of NaCl in 5 mL of water and stir thoroughly. Cover the beaker with plastic wrap, leaving a small hole for water evaporation. Place in a fume hood. After 48 hours, remove the precipitated crystals, dry them, and set aside.

[0042] 3.2 Grind 5 g of melamine and 2 g of the crystals obtained in step 3.1 for 5 hours, mix well and form a fine powder for later use;

[0043] 3.3 Take 6 g of the fine powder from step 3.2 and treat it at 650°C under argon atmosphere for 2 hours. Then wash it with water 3 times and ethanol 3 times, and dry it for later use.

[0044] 3.4 Take 5 g of the sample from step 3.3, add 0.35 g of thioglycolic acid, and grind for 8 hours to mix evenly. After treating at 400°C under argon atmosphere for 1 hour, wash with water and ethanol three times each. Then, ultrasonically disperse the sample in water at a mass ratio of sample to water of 20%:1 to obtain a suspension.

[0045] 3.5 Take the suspension sample from step 3.4 and centrifuge it at 3500 rpm for 30 minutes. The upper suspension is the prepared fluorescent probe. The luminescence color and microstructure of the resulting sample are similar to those in Example 1, and the sample exhibits bright green luminescence.

[0046] Comparative Example 1

[0047] 1.1 Grind 0.04 g chloroauric acid, 2 g NaCl and 5 g melamine for 5 hours, mix well and form a fine powder for later use;

[0048] 1.2 Take 6 g of the fine powder from step 1.1 and treat it at 600°C under argon atmosphere for 3 hours. Then wash it with water 3 times and ethanol 3 times, and dry it for later use.

[0049] 1.3 Take 5 g of the sample from step 1.2, add 0.35 g of thioglycolic acid, and grind for 6 hours to mix evenly. After treating at 350 degrees Celsius under an argon atmosphere for 1.5 hours, wash with water and ethanol three times each. Then, ultrasonically treat the sample at a mass ratio of sample to water of 35%:1. Although the obtained sample is in the form of nanosheets, the luminescence brightness is very low and does not meet the requirements of a fluorescent probe.

[0050] Comparative Example 2

[0051] The other steps are the same as those in Example 1, except that step 1.3 is heat-treated in an air atmosphere. The obtained sample does not emit light and does not have the properties of a fluorescent probe.

[0052] Comparative Example 3

[0053] The other steps were the same as those in Example 1, except that chloroauric acid was not added in step 1.1. The obtained sample emitted weak blue luminescence under ultraviolet light, and the brightness was very low.

[0054] Comparative Example 4

[0055] The other steps were the same as in Example 1, except that the grinding time in step 1.2 was 3 hours. The obtained sample showed only a weak blue light under ultraviolet light, which was not bright enough to meet the probe requirements.

[0056] Comparative Example 5

[0057] The other steps were the same as in Example 1, except that the heat treatment temperature in step 1.3 was 450 degrees Celsius. The resulting sample showed only a faint green light under ultraviolet light, which was not bright enough to meet the probe requirements.

[0058] Comparative Example 6

[0059] The remaining steps were the same as in Example 1, except that 0.15 g of chloroauric acid was added. The results showed that the chloroauric acid crystallized on its own and did not completely enter the sodium chloride crystals. The final sample showed weak defective blue light under ultraviolet light, which did not meet the probe luminescence color and brightness requirements.

Claims

1. A method for preparing a fungal fluorescent probe, characterized in that The following steps are involved: (1) Dissolve chloroauric acid and sodium chloride in water, then ventilate and let stand until crystals are completely precipitated to obtain sodium chloride crystals containing chloroauric acid; (2) mixing sodium chloride crystals containing chloroauric acid and a graphite-phase carbon nitride precursor and performing a grinding pretreatment; (3) The mixture obtained after grinding is heated to perform a first heat treatment to obtain layered g-C3N4 nanosheets embedded with gold nanoparticles; (4) The layered g-C3N4 nanosheets embedded with gold nanoparticles are mixed with an organic acid containing a thiol group, subjected to a grinding pretreatment, and then subjected to a second heat treatment by heating; (5) Washing the sample after the second heat treatment, and then ultrasonically dispersing it in water, centrifuging it after ultrasonication to obtain the suspension and obtain the fungal fluorescent probe; In step (1), the mass ratio of chloroauric acid to sodium chloride is 0.01-0.02:1, and the amount of water used ensures that the sodium chloride reaches a saturated concentration; In step (2), the mass ratio of the graphite phase carbon nitride precursor to the sodium chloride crystal containing chloroauric acid is 1-2:0.3-0.5, and the grinding time is 5-10 hours; In step (3), the first heat treatment is carried out in an inert atmosphere at a temperature of 550-650 degrees Celsius for 2-4 hours; In step (4), the mass ratio of the organic acid containing thiol groups to the layered g-C3N4 nanosheets embedded with gold nanoparticles is 0.05-0.1:1, and the grinding time is 4-8 hours; In step (4), the second heat treatment is carried out in an inert atmosphere at a temperature of 300-400 degrees Celsius and a treatment time of 1-2 hours.

2. The preparation method according to claim 1, wherein: The graphite phase carbon nitride precursor is melamine or dicyandiamide.

3. The preparation method according to claim 1, wherein: The ratio of sodium chloride to water in step (1) is 2g:5-6ml.

4. The preparation method according to claim 1, wherein: In step (4), the organic acid containing a thiol group is thioglycolic acid or thiopropionic acid.

5. A fungal fluorescent probe prepared according to the method for preparing a fungal fluorescent probe according to any one of claims 1 to 4.

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