Ultraviolet quenching antagonist for enhancing initial fluorescence intensity and preparation method thereof

Abstract

The invention discloses an ultraviolet quenching antagonist for enhancing initial fluorescence intensity and a preparation method thereof, and is characterized in that the ultraviolet quenching antagonist is formed by mixing a compound for enhancing the stability of a fluorophore, a compound for improving the initial fluorescence brightness of the fluorophore and a base solution; the compound for enhancing the stability of the fluorophore is n-propyl gallate, 1, 4-diazabicyclo [2, 2, 2] octane or tertiary butyl hydroquinone; the compound capable of improving the initial fluorescence brightness of the fluorophore is trans-1, 2-stilbene, oxidized resveratrol or 1, 6-diphenyl-1, 3, 5-hexatriene, and the compound capable of improving the initial fluorescence brightness of the fluorophore is a compound capable of improving the initial fluorescence brightness of the fluorophore. The composition is high in energy transfer speed, the initial fluorescent brightness of common fluorophores can be remarkably improved, and most fluorophores can maintain higher initial fluorescent brightness.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescence microscopy imaging technology, in particular to the field of fluorescence in situ hybridization (FISH) and immunofluorescence (IF), and more particularly to the field of fluorescence imaging technology for improving the quality of fluorescence imaging, especially for low-abundance targets. BACKGROUND

[0002] The initial fluorescence intensity is used to represent the relative brightness of the fluorescent group when it is excited, which is determined by the extinction coefficient (also known as the absorption coefficient, ε) and the fluorescence quantum yield Φ, where the extinction coefficient ε is the intensity of the fluorescent molecule absorbing light of a specific wavelength; the fluorescence quantum yield Φ is the efficiency of converting absorbed light into emitted light. Generally, the initial fluorescence intensity is represented as the product of the values of the two. Under certain experimental conditions, these two parameters are characteristic constants for each individual fluorescent group, and the higher the values of the two parameters, the higher the fluorescence intensity. In fluorescence microscopy imaging technology, fluorescent groups with higher initial fluorescence intensity are more conducive to improving the quality of imaging and are more suitable for detecting lower abundance targets.

[0003] The size of the extinction coefficient ε is related to the properties of the fluorescent group, the properties of the solvent, and the wavelength of the excitation light. In fluorescence microscopy imaging technology, for a specific fluorescent group under a specific wavelength of excitation light, its extinction coefficient ε is related to the properties of the solvent. In fluorescence microscopy imaging, fluorescence quenching antagonists are often used to enhance the stability of fluorescent groups to delay the fluorescence quenching (photobleaching) phenomenon in imaging. However, the addition of most quenching antagonists will reduce the initial fluorescence intensity, which may be due to the decrease in the extinction coefficient ε of the fluorescent group in the corresponding quenching antagonist. However, in practical applications, the use of quenching antagonists is still a better strategy, as the amount of initial fluorescence intensity reduction is within an acceptable range, maintaining the stability of the fluorescent group is more conducive to meeting the imaging needs, especially in experiments with high anti-quenching requirements.

[0004] The decay process of the excited-state fluorescent molecule includes radiative transition and non-radiative transition, so the fluorescence quantum yield can also be represented as Y f =k f / (k f +∑K), where Y f represents the fluorescence quantum yield, k f represents the rate constant of fluorescence emission, and ∑K represents the sum of the rate constants of various intramolecular non-radiative decay processes. It can be seen that the size of the fluorescence quantum yield depends on the competition result of the fluorescence emission process and the non-radiative transition process. If the rate of non-radiative transition is much smaller than the rate of radiative transition, i.e., ∑K << k f , the value of Y f is close to 1. Under normal circumstances, Y fThe value of the fluorescence quantum yield is always less than 1, and the closer it is to 1, the higher the fluorescence brightness. The size of the fluorescence quantum yield is related to the structure and properties of the fluorescent group, and is mainly related to the environmental factors. The method of changing the solution environment to improve the fluorescence quantum yield can be integrated into the research and development of fluorescence quenching antagonists to achieve stable fluorescence groups while maintaining high initial fluorescence brightness.

[0005] Luis A. Campos et al. found that the combination of water-soluble vitamin E (Trolox) and β-mercaptoethylamine (β-MEA) can achieve microsecond resolution on short typical double-stranded DNA (B-DNA) molecules and significantly improve the fluorescence quantum yield of the fluorescent group Alexa Fluor 594. Widengren found that the antioxidant properties of KI can enhance fluorescence brightness. Gonzalo Cosa et al. found that the presence of Ni 2+ can increase the number of light quantum outputs of Alexa Fluor 647 by 25 times, but high concentrations of Ni 2+ have cytotoxicity and are difficult to apply to biological imaging. Olivier added cyclooctatetraene (COT) in the buffer system of super-resolution microscopy technology (STORM) to significantly improve the fluorescence quantum yield of Alexa Fluor 647. But the effect of COT on light stability varies with the type of fluorescent molecule, such as Cy3B and Cy5, ATTO 655, rhodamine and fluorescein, which have different competitive electron transfer processes (intramolecular or / and intermolecular electron movement). In general, the energy transfer rate is faster than the electron transfer rate, which can improve the light stability and facilitate the increase of the radiation transition rate. SUMMARY

[0006] The purpose of the present application is to provide a UV quenching antagonist for enhancing the initial fluorescence intensity and a preparation method thereof.

[0007] The purpose of the present application is achieved by a UV quenching antagonist for enhancing the initial fluorescence intensity, characterized by being mixed by a compound for enhancing the stability of the fluorescent group, a compound for improving the initial fluorescence brightness of the fluorescent group, and a base solution; the compound for enhancing the stability of the fluorescent group is n-propyl gallate, 1,4-diazabicyclo[2,2,2]octane or tert-butyl hydroquinone; and the compound for improving the initial fluorescence brightness of the fluorescent group is trans-1,2-stilbene, oxidized tyrosol or 1,6-diphenyl-1,3,5-hexatriene.

[0008] The object of the present application can also be achieved in that the compound for enhancing the stability of the fluorescent group is tert-butyl hydroquinone, the compound for improving the initial fluorescence brightness of the fluorescent group is 1,6-diphenyl-1,3,5-hexatriene, and the base solution is 80% glycerol-PBS.

[0009] The concentration of the tert-butyl hydroquinone is 0.2% to 1.0% w / v, and the concentration of the 1,6-diphenyl-1,3,5-hexatriene is 0.2 to 1.5 µM.

[0010] A preparation method of an ultraviolet quenching antagonist for enhancing initial fluorescence intensity, characterized by two steps of base solution preparation and two-component mixing of tert-butyl hydroquinone and 1,6-diphenyl-1,3,5-hexatriene. The base solution 80% glycerol-PBS is prepared from the following volume fractions of raw materials: glycerol 8.0 parts, 10×PBS of 100 mM, pH=7.0 1.0 part, and ddH2O 1.0 part. The two-component mixing is carried out from the following volume fractions of raw materials and steps: 0.10 to 0.50 parts of a tert-butyl hydroquinone stock solution with a concentration of 20% w / v and a solvent of dimethyl sulfoxide are added to 9.35 to 9.88 parts of the 80% glycerol-PBS base solution and mixed thoroughly; then 0.02 to 0.15 parts of a 1,6-diphenyl-1,3,5-hexatriene stock solution with a concentration of 100 µM and a solvent of dimethyl sulfoxide are added and mixed thoroughly; a quenching antagonist with a final concentration of 0.2% to 1.0% (w / v) of tert-butyl hydroquinone and a final concentration of 0.2 to 1.5 µM of 1,6-diphenyl-1,3,5-hexatriene is obtained.

[0011] The two-component mixing is carried out from the following volume fractions of raw materials and steps: 0.10 to 0.50 parts of a tert-butyl hydroquinone stock solution with a concentration of 20% w / v and a solvent of dimethyl sulfoxide are added to 9.35 to 9.88 parts of the 80% glycerol-PBS base solution and mixed thoroughly; then 0.02 to 0.15 parts of a 1,6-diphenyl-1,3,5-hexatriene stock solution with a concentration of 100 µM and a solvent of dimethyl sulfoxide are added and mixed thoroughly; a quenching antagonist with a final concentration of 0.2% to 1.0% (w / v) of tert-butyl hydroquinone and a final concentration of 0.2 to 1.5 µM of 1,6-diphenyl-1,3,5-hexatriene is obtained.

[0012] The present application has the following beneficial effects: compared with the prior art, the present application is a composition with faster energy transfer speed, which can significantly improve the initial fluorescence brightness of common fluorescent groups and enable most fluorescent groups to maintain higher initial fluorescence brightness.

[0013] Further research shows that TBHQ and DPH have a synergistic effect on enhancing the stability of the fluorescent group and improving the initial fluorescence brightness performance. TBHQ alone can only enhance the stability of the fluorescent group, and DPH alone has no beneficial effect (its anti-quenching effect is weak, and the fluorescence quenching is too fast to affect the observation effect), and only when the two act together can both enhance the stability of the fluorescent group and improve the initial fluorescence brightness. The DPH has the characteristics of trace (micromolar level) and high efficiency in use. In the present application, TBHQ and DPH are determined as a combination, which is beneficial to improve the fluorescence microscopic imaging quality.

[0014] The anti-quenching effect and initial fluorescence brightness of the quenching antagonist of the present application and Vectashield (a commercial quenching antagonist) in 4', 6-diamidino-2-phenylindole (DAPI), Hoechst 33342, SYTO-80, Cy3, carboxyfluorescein (FAM) and SpectrumGreen were compared, and the results showed that in terms of anti-quenching, the quenching antagonist of the present application was superior to Vectashield in Hoechst 33342, SYTO-80 and SpectrumGreen; in terms of initial fluorescence brightness, the quenching antagonist of the present application was superior to Vectashield in DAPI, Hoechst 33342, SYTO-80, Cy3 and SpectrumGreen. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is an anti-quenching effect diagram of part of the quenching antagonists involved in the present application. The diagram shows that the human peripheral blood cells are re-stained with Hoechst 33342 under the fluorescence microscope 40x ocular lens, and the image is collected after continuous irradiation for 2 minutes; ET: exposure time.

[0016] Figure 2 It is a statistical comparison diagram of tert-butylhydroquinone (TBHQ) and 1,6-diphenyl-1,3,5-hexatriene (DPH) combination in improving the initial fluorescence brightness performance (the fluorescent group is the fluorescent group labeled in Ampirin GSP 21q Probe). A is the fluorescence intensity trend; B is the fluorescence decay rate; C is the initial fluorescence brightness (the exposure time when the image is collected is used as the statistical quantity). The statistical analysis method in the diagram is Duncan's new multiple range test, and different lowercase letters represent P <0.05 level difference is significant.

[0017] Figure 3 This is a statistical graph comparing the effects of the quenching antagonist and Vectashield on different fluorescent groups in this invention. A represents the fluorescence decay rate; B represents the initial fluorescence intensity (based on the exposure time at image acquisition). The statistical analysis method used in the graph is a multiple t-test. P <0.05、 P <0.01 and P The difference was not statistically significant at a level of <0.001. Detailed Implementation

[0018] This invention discloses the application of a class of compounds in improving initial fluorescence brightness. The composition thereof serves as a fluorescence quenching antagonist, which enhances the stability of fluorescent groups and improves initial fluorescence brightness.

[0019] The compound that enhances the stability of the fluorescent group in this invention is one of n-propyl gallate (NPG), 1,4-diazabicyclo[2,2,2]octane (DABCO), and tert-Butylhydroquinone (TBHQ).

[0020] The compound used in this invention to enhance the initial fluorescence brightness of the fluorescent group is one of trans-stilbene (tSB), oxyresveratrol (OR), and 1,6-diphenyl-1,3,5-hexatriene (DPH).

[0021] Furthermore, the compound that enhances the stability of the fluorescent group is preferably TBHQ.

[0022] Furthermore, the compound that enhances the initial fluorescence brightness of the fluorescent group is preferably DPH.

[0023] This invention provides a fluorescence quenching antagonist with initial fluorescence brightness enhancement properties and its preparation method.

[0024] The fluorescence quenching antagonist prepared in this invention is a colorless liquid formulation, using 80% glycerol as the basic medium. 80% glycerol satisfies the refractive index requirement in fluorescence microscopy (the refractive index of 80% glycerol is approximately 1.45) and also provides a formulation with relatively low viscosity, facilitating pipetting; furthermore, glycerol is colorless, odorless, safe, and non-toxic, making it an environmentally friendly solvent.

[0025] The fluorescence quenching antagonist is buffered in phosphate-buffered saline (PBS) with a pH of 7.0. PBS is the most widely used buffer in biochemistry and cell biology, providing a relatively stable pH and ionic strength system in cell or tissue samples. pH 7.0 ensures solute stability and is compatible with most fluorescent groups, especially some novel ones.

[0026] The first component of the fluorescence quenching antagonist is tert-butylhydroquinone (TBHQ), which is relatively safe and poses no health hazard to operators. At a concentration of 0.2% to 1.0% (w / v), it can be well dissolved in glycerol to form a stable solution. TBHQ has strong antioxidant properties and can form quinones upon oxidation to form the TBHQ-Quinone reduction oxidation system (ROXS), which provides photoprotection for fluorescent groups.

[0027] The second component of the fluorescence quenching antagonist is 1,6-diphenyl-1,3,5-hexanetriene (DPH) at a concentration of 0.2–1.5 µM. Unbound by theoretical constraints, 1,6-diphenyl-1,3,5-hexanetriene can undergo rapid energy transfer, thereby directly reducing the non-radiative transition rate, increasing the radiative transition rate, and enhancing the initial fluorescence brightness.

[0028] The preparation method of the fluorescence quenching antagonist is divided into two steps: base liquid preparation and component mixing.

[0029] The base solution is 80% glycerol-PBS (pH=7.0), prepared from the following parts by volume of raw materials: 8.0 parts of glycerol; 1.0 part of 10×PBS (100 mM, pH=7.0) buffer; and 1.0 part of ddH2O.

[0030] The two components are prepared by mixing the following parts by volume of raw materials and steps: 0.10-0.50 parts of TBHQ (20% w / v, solvent dimethyl sulfoxide, DMSO) stock solution are added to 9.35-9.88 parts of 80% glycerol-PBS (pH=7.0) base solution and mixed thoroughly; then 0.02-0.15 parts of DPH (100 µM, DMSO) stock solution are added and mixed thoroughly to obtain the fluorescence quenching antagonist.

[0031] Under the action of the initial fluorescence brightness-enhancing quencher antagonist described in this invention, the initial fluorescence brightness of most fluorescent groups is significantly enhanced. The quencher antagonist disclosed in this invention was compared with Vectashield (a commercially available quencher antagonist) in DAPI, Hoechst 33342, SYTO-80, Cy3, FAM, and SpectrumGreen in terms of anti-quenching effect and initial fluorescence brightness. The results show that in terms of anti-quenching, the quencher antagonist described in this invention is superior to Vectashield in Hoechst 33342, SYTO-80, and SpectrumGreen; in terms of initial fluorescence brightness, the quencher antagonist described in this invention is superior to Vectashield in DAPI, Hoechst 33342, SYTO-80, Cy3, and SpectrumGreen.

[0032] Compared with the prior art, the fluorescence quenching antagonist described in this invention can maintain higher initial fluorescence brightness for most commonly used fluorescent groups.

[0033] Example 1: Preparation of the initial fluorescence brightness-enhancing fluorescence quenching antagonist of the present invention.

[0034] The formulation of this invention is composed of two compounds: tert-Butylhydroquinone (TBHQ) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The specific preparation method of the quenching antagonist is as follows: 1. Preparation of mother liquor (1) TBHQ (20% w / v, DMSO) ① Raw materials: Aladdin B105351, Lot#D2114321, 98.37% GC, Mw=166.22 g / mol, CAS: 1948-33-0.

[0035] ② Preparation: Weigh 0.5000 g TBHQ and add 2.5 ml DMSO. Mix thoroughly and store at 4°C away from light for later use.

[0036] (2) DPH (100 µM, DMSO) ① Raw materials: Aladdin D105558, Lot#J2129526, 98.20% HPLC, Mw=232.32 g / mol, CAS: 1720-32-7.

[0037] ②Preparation: a. Weigh 0.0028 g DPH and add it to 4.857 ml DMSO. Incubate in a 70℃ water bath for 10 min and mix thoroughly to prepare a 3 mM stock solution. b. Take 90 µl of 3 mM stock solution and add 2610 µl of DMSO. Mix thoroughly and store at 4°C in the dark for later use.

[0038] (3) 80% glycerol PBS (pH=7.0) ①Ingredients: a. Glycerol: Aladdin G116210, Lot#G2108205, 99.50% GC, Mw=92.09 g / mol, CAS: 56-81-5; b.10×PBS: Sigma P5493, Lot#SLBX1608, pH=7.0, 100 mM; c.ddH2O: Water from the pure water system is filtered through a 0.22 µm filter.

[0039] ② Preparation (100 ml): Measure 80 ml of glycerol, add 10 ml of 10×PBS, then add 10 ml of ddH2O, mix thoroughly, centrifuge to remove air bubbles, and store at 4℃ for later use.

[0040] 2. Formulation of antagonist quenching agents Mix thoroughly according to the dosage in the table below.

[0041]

[0042] Example 2: Comparison of the anti-quenching effect and initial fluorescence intensity of the quenching antagonist of the present invention with Vectashield in DAPI, Hochst 33342 and SYTO-80.

[0043] Based on fluorescence in situ hybridization (FISH) technology, fluorescence intensity changes are acquired through fluorescence microscopy. The fluorescence intensity of these images is then quantitatively analyzed using ImageJ software, ultimately generating visualized data on the anti-quenching effect. The specific implementation method is as follows: 1. Experimental Methods (1) Preparation of cell droplets ① Take 10 μl of Carnot fixative to fix the cells and drop them onto a glass slide in the air. Let them air dry at room temperature and mark the cell drop positions with a diamond pen. ② Bake the slices at 60℃ for 1 hour; ③ Wash the slide twice in 2× sodium citrate solution (SSC) at room temperature, 3 min each time; ④ Transfer the slide to ethanol for gradient dehydration (70%, 85%, 100%), 1 min / bottle, and air dry at room temperature.

[0044] (2) Cells were counterstained with fluorescent dyes ① The fluorescent dye DAPI (Yeasen 40728ES03, Lot#D8013020, 10 mg / ml) was diluted to a working concentration of 1 μg / ml using Vectashield and the formulation prepared in Example 1, respectively; the fluorescent dye Hoechst 33342 (Sigma B2261, Lot#117M4047V, 10 mg / ml) was diluted to a working concentration of 1 μg / ml using Vectashield and the formulation prepared in Example 1, respectively; the fluorescent dye SYTO-80 (Invitrogen S11360, Lot#2049035, 5 mM) was diluted 10× to a working concentration using Vectashield and the formulation prepared in Example 1, respectively, to obtain a mixture of 6 fluorescent dyes and quenching antagonists; ② Take 5 μl of the mixture of fluorescent dye and quenching antagonist and add it to the marked cell area on the glass slide, then cover with a coverslip.

[0045] (3) Observe and collect images under a microscope Select a region of cells and take one image every 30 seconds (record the exposure time of the first image). Take 5 images per field of view (total 2 minutes). Change fields of view and continue taking images, keeping the shooting parameters constant. Take 3 images per sample. Field of view selection principles: a. The concentration of the staining agent should not be too high, and the background should be clear; b. The cell nucleus outline is clear and has a high contrast with the background; c. The difference in brightness between cell nuclei should not be too great; d. The number of cells in the field of view should not be too high (no more than 10).

[0046] (4) Statistical analysis of quenching resistance effect After the images were captured, ImageJ was used to analyze the average fluorescence intensity in each field of view [average fluorescence intensity = total fluorescence intensity of the region / area of ​​the region at 0 sec], and the fluorescence decay rate in the field of view under the action of each quenching antagonist was calculated based on the average fluorescence intensity corresponding to the total irradiation time.

[0047] 2. Experimental Results The results show that smaller values ​​correspond to stronger effects. As shown in Table 1, except for DAPI, the anti-quenching effect of the quenching antagonists of this invention is comparable to or slightly stronger than that of Vectashield. The initial fluorescence brightness is higher than that of Vectashield in all cases, and significantly higher than Vectashield in two (2 / 3) of the results.

[0048] Table 1. Comparison of anti-quenching effect and initial fluorescence intensity in nucleic acid fluorescent dye experiments (±SD, n=3)

[0049] Note: The statistical analysis method in the table is a multiple t-test; different lowercase letters indicate different values. P The difference was statistically significant at levels <0.05.

[0050] Example 3: Comparison of the anti-quenching effect and initial fluorescence intensity of the quenching antagonist of the present invention and Vectashield in FAM, Cy3 and SpectrumGreen labeled probes.

[0051] 1. Experimental Methods (1) Preparation of cell droplets ① Take 10 μl of Carnot fixative to fix the cells and drop them onto a glass slide in the air. Let them air dry at room temperature and mark the cell drop positions with a diamond pen. ② Bake the slices at 60℃ for 1 hour; ③ Wash the slide twice in 2×SSC solution at room temperature, 3 min each time; ④ Transfer the slide to ethanol for gradient dehydration (70%, 85%, 100%), 1 min / bottle, and air dry at room temperature.

[0052] (2) Fluorescence in situ hybridization (FISH) ① Take FAM-labeled probe (FAM-NHS: Aladdin C107881, Lot# D1820039; the probe was prepared by inserting AA-dUTP into the target fragment by PCR and then conjugating it with FAM-NHS), Cy3-labeled probe (Cy3-dUTP: APExBIOB8202, Lot# B82021133C979; the probe was prepared by PCR) and CEP Y SpectrumGreen probe (Vysis7J20-50, Lot# 511210) and hybridization solution at a ratio of 1:3 (total 4 μl), mix well, drop onto the surface of a glass slide, cover with a coverslip, and seal with cement glue; ② Place the slide in a hybridization apparatus and incubate at 82 ℃ for 10 min, then hybridize at 42 ℃ for 2 h; ③ Remove the slide, remove the coverslip, and immerse it in 2×SSC solution preheated to 65℃ for 10 min; ④ Transfer the slide to 2×SSC solution at room temperature and wash for 3 min; ⑤ Transfer the glass slide to ethanol for gradient dehydration (70%, 85%, 100%), 1 min / bottle, and air dry at room temperature; ⑥ Add 5 μl each of Vectashield and the preparation from Example 1 to the hybridization area and cover with a coverslip.

[0053] (3) Observe and collect images under a microscope Select areas with clear fluorescence signals and take one image every 30 seconds (record the exposure time of the first image). Take a total of 5 images per field of view (2 minutes in total). Change fields of view and continue taking images, keeping the shooting parameters constant. Take 3 images per sample. Field of view selection principles: a. The hybridization signal is bright, with clear outlines and high contrast with the background; b. The difference in the size and brightness of the hybridization signal should not be too large; c. There should not be too many hybridization signals in the field of view (no more than 10).

[0054] (4) Statistical analysis of quenching resistance effect After the images were captured, ImageJ was used to analyze the average fluorescence intensity in each field of view [average fluorescence intensity = total fluorescence intensity of the region / area of ​​the region at 0 sec], and the fluorescence decay rate in the field of view under the action of each quenching antagonist was calculated based on the average fluorescence intensity corresponding to the total irradiation time.

[0055] 2. Experimental Results The smaller the value shown in the results, the stronger the corresponding effect. As shown in Table 2, except for FAM, the anti-quenching effect of the quenching antagonist of this invention is comparable to or slightly stronger than that of Vectashield. The initial fluorescence brightness is also higher than Vectashield except for FAM, and significantly higher than Vectashield in SpectrumGreen. The quenching antagonist described in this invention also exhibits an anti-quenching effect with enhanced initial fluorescence brightness on fluorescently labeled probes.

[0056] Table 2 Comparison of quenching resistance and initial fluorescence intensity in fluorescently labeled probe experiments (±SD, n=3)

[0057] Note: The statistical analysis method in the table is a multiple t-test; different lowercase letters indicate different values. P The difference was statistically significant at levels <0.05.

Claims

1. A UV quenching antagonist that enhances the intensity of initial fluorescence, characterized in that, It is composed of a compound that enhances the stability of the fluorescent group, a compound that improves the initial fluorescence brightness of the fluorescent group, and a base liquid; the compound that enhances the stability of the fluorescent group is propyl gallate, 1,4-diazabicyclo[2,2,2]octane, or tert-butylhydroquinone; the compound that improves the initial fluorescence brightness of the fluorescent group is trans-1,2-stilbene, resveratrol oxide, or 1,6-diphenyl-1,3,5-hextriene.

2. The ultraviolet quenching antagonist that enhances the intensity of initial fluorescence according to claim 1, characterized in that, The compound that enhances the stability of the fluorescent group is tert-butylhydroquinone, the compound that improves the initial fluorescence brightness of the fluorescent group is 1,6-diphenyl-1,3,5-hexanetriene, and the base solution is 80% glycerol-PBS.

3. The ultraviolet quenching antagonist that enhances the intensity of initial fluorescence according to claim 2, characterized in that, The concentration of tert-butylhydroquinone is 0.2% to 1.0% w / v; the concentration of 1,6-diphenyl-1,3,5-hextriene is 0.2 to 1.5 µM.

4. A method for preparing an ultraviolet quenching antagonist that enhances the intensity of initial fluorescence, characterized in that, The process consists of two steps: preparation of the base solution and mixing of two components: tert-butylhydroquinone (80% glycerol-PBS). The base solution, 80% glycerol-PBS, is prepared from the following parts by volume: 8.0 parts glycerol; 1.0 part 100 mM, pH 7.0 10×PBS; 1.0 part ddH2O. The mixing of the two components is carried out using the following parts by volume and steps: 0.10–0.50 parts of 20% w / v tert-butylhydroquinone stock solution in dimethyl sulfoxide solvent are added to 9.35–9.88 parts of the 80% glycerol-PBS base solution and thoroughly mixed; then 0.02–0.15 parts of 100% glycerol-PBS are added. The mixture is thoroughly mixed in a mother liquor of 1,6-diphenyl-1,3,5-hextriene in dimethyl sulfoxide (µM) to obtain a quenching antagonist with a final concentration of 0.2%–1.0% (w / v) of tert-butylhydroquinone and a final concentration of 0.2–1.5 µM of 1,6-diphenyl-1,3,5-hextriene.

5. The method for preparing the ultraviolet quenching antagonist that enhances the initial fluorescence intensity according to claim 4, characterized in that, The two-component mixture is prepared by mixing the following volume percentages: 2.50% (by volume) of tert-butylhydroquinone stock solution at a concentration of 20% w / v and in dimethyl sulfoxide solvent; 1.00% (by volume) of 1,6-diphenyl-1,3,5-hextriene stock solution at a concentration of 100 µM and in dimethyl sulfoxide solvent; and 96.50% (by volume) of 80% glycerol-PBS. This yields a quenching antagonist with a final concentration of 0.5% (w / v) of tert-butylhydroquinone and a final concentration of 1.0 µM of 1,6-diphenyl-1,3,5-hextriene.

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