Reversible regulation fluorescent gold nanoclusters and preparation method thereof

Abstract

The application is suitable for the technical field of nanocluster preparation, and provides reversible control fluorescent gold nanoclusters and a preparation method thereof, which comprises the following steps: adding 4mM chloroauric acid solution 221µL into a CK solution with a concentration of 2mM 443µL after being fully dissolved, then adding 2M NaOH 14.7µL, adjusting the pH value of the solution to 13, immediately mixing, wrapping with tin paper, transferring to a shaking incubator for reaction for 10h, and obtaining H-AuNCs with bright red fluorescence. 60µM H-AuNCs are co-granted with HUVEC cells for 2h, and bright red fluorescence is formed in the cells, so that biological imaging can be realized. The powder obtained by drying the H-AuNCs is transferred into a dimethyl sulfoxide solution to obtain organic phase D-AuNCs with bright blue fluorescence. The application prepares CK-AuNCs with high fluorescence intensity, and can realize reversible adjustment of the fluorescence color.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanocluster preparation, and particularly relates to a reversible fluorescence gold nanocluster and a preparation method thereof. BACKGROUND

[0002] The fluorescence adjustment of AuNCs mainly focuses on the adjustment of fluorescence intensity and color. The fluorescence intensity can be adjusted through various methods such as aggregation-induced emission, aggregation-induced quenching, ion doping and self-assembly. The adjustment of fluorescence color is mainly carried out in terms of gold core structure and quantity, ligand coverage and local action, organic molecular ligand and electron transfer between ligand and gold core and assembly structure, and the related research is less. Most of the current reports are one-way fluorescence color adjustment, and the reversible adjustment of double fluorescence of AuNCs is still less. Therefore, we propose a reversible fluorescence gold nanocluster and a preparation method thereof. SUMMARY

[0003] The application aims to provide a reversible fluorescence gold nanocluster and a preparation method thereof, and aims to solve the problems proposed in the background.

[0004] To achieve the above-mentioned purpose, the application provides the following technical scheme.

[0005] A preparation method of a reversible fluorescence gold nanocluster, comprising the following steps:

[0006] In an aqueous solution, CK is used as a template to react with chloroauric acid under alkaline conditions to synthesize H-AuNCs with bright red fluorescence, and biological imaging is realized.

[0007] Further, the specific operation of configuring the CK solution with a concentration of 2 mM is as follows: 1.5 mg of CK is dissolved in 443 μL of distilled water to obtain a CK solution with a concentration of 2 mM.

[0008] Further, the preparation method of the gold nanocluster comprises the following steps:

[0009] After the 443 μL of CK solution with a concentration of 2 mM is fully dissolved, 221 μL of 4 mM chloroauric acid solution is added, followed by the addition of 14.7 μL of 2 M NaOH, and the pH value of the solution is adjusted to 13. Immediately mix, wrap with tin paper, and transfer to a shaking incubator for reaction for 10 h to obtain H-AuNCs with bright red fluorescence.

[0010] Further, the temperature of the shaking incubator is 37℃, and the rotation speed is 180 rpm.

[0011] Further, the specific operation of adjusting the fluorescence of the H-AuNCs is as follows:

[0012] 200 µL H-AuNCs were dried by using a vacuum freeze-dryer for 12h, and the freeze-dried H-AuNCs powder was taken in 200 µL DMSO and placed in a shaking incubator to prepare D-AuNCs with bright blue fluorescence, which was taken out every day to measure the fluorescence, and the conversion of H-AuNCs to D-AuNCs within 7 days was monitored.

[0013] Further, the D-AuNCs were freeze-dried in a freeze-dryer to obtain solid-state D-AuNCs, which were introduced into an aqueous solution, and the fluorescence changed from blue to red; the D-AuNCs solution was taken, and an aqueous solution was added, and the fluorescence changed from blue to red.

[0014] A reversible fluorescence gold nanocluster preparation method.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] (1) A polypeptide containing a gold cluster stable reduction region, a flexible Linker region and a conformational regulation region is designed by using a modular strategy, and H-AuNCs with bright red fluorescence are successfully prepared in an aqueous solution by using the polypeptide as a template, and can be used for biological imaging.

[0017] (2) The conformational change of the polypeptide ligand on the surface of AuNCs is induced by solvent change to realize the reversible regulation of the double fluorescence colors. 200 µL H-AuNCs are freeze-dried and added to 200 µL DMSO, and then converted into D-AuNCs, which can change from bright red fluorescence to bright blue fluorescence within seven days, and the fluorescence change is linear with time. The D-AuNCs are freeze-dried, and an aqueous solution is added dropwise, and the fluorescence changes from blue to red within 5s; an aqueous solution is added dropwise in the D-AuNCs solution, and the fluorescence changes from blue to red within 2min. In addition, the relationship between the fluorescence change of D-AuNCs and the water content is determined. The solvent-responsive change of D-AuNCs is linear within a certain range.

[0018] (3) The reversible conversion of AuNCs from red fluorescence to blue fluorescence is realized by regulating H2O and DMSO solvents, and the reason is that the solution change between H2O and DMSO can induce the conformational change of the polypeptide ligand on the surface of AuNCs. The fluorescence of D-AuNCs is increased by three times by modifying the benzene ring on the side chain of the polypeptide CK on the surface of D-AuNCs, which indicates that the increase of π-π conjugation between polypeptides leads to the dramatic increase of fluorescence. This mechanism guides the design of environment-responsive fluorescent materials. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1In the figure, A is the fluorescence color change diagram of H-AuNCs; B is the fluorescence conversion spectrum of H-AuNCs; C is the fluorescence color change-time relationship diagram of H-AuNCs; D is the CIE color model.

[0020] Figure 2 In the figure, A is the fluorescence color change diagram of D-AuNCs; B is the fluorescence conversion spectrum of D-AuNCs; C is the fluorescence color change-time relationship diagram of D-AuNCs; D is the CIE color model.

[0021] Figure 3 It is the fluorescence spectrum of D-AuNCs added with different types of organic solvents.

[0022] Figure 4 It is the fluorescence spectrum of D-AuNCs added with different alcohols.

[0023] Figure 5 It is the fluorescence titration spectrum of Tyr.

[0024] Figure 6 In the figure, A is the hydrate particle size of benzaldehyde modified D-AuNCs; B is the fluorescence conversion spectrum of benzaldehyde modified D-AuNCs; C is the hydrate particle size of acetic anhydride modified D-AuNCs; D is the fluorescence conversion spectrum of acetic anhydride modified D-AuNCs.

[0025] Figure 7 In the figure, A is the fluorescence reverse regulation diagram of solid powder D-AuNCs; B is the fluorescence reverse regulation diagram of liquid D-AuNCs. Embodiment

[0026] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0027] The specific implementation of the present application is described in detail below in combination with specific examples.

[0028] An embodiment of the present application provides a preparation method of reversibly regulated fluorescent gold nanoclusters, comprising the following steps:

[0029] H-AuNCs with bright red fluorescence are synthesized by reacting CK as a template with chloroauric acid in an aqueous solution under alkaline conditions, and biological imaging is realized.

[0030] In the embodiment of the present application, the polypeptide ligand with both stability and reducing properties, i.e., the polypeptide containing cysteine capable of forming Au-S, tyrosine providing reducing power, and aromatic amino acids and positively charged amino acids arranged alternately, has a sequence of CCYGGYRFKYRFK (abbreviated as CK). The present application uses the polypeptide (abbreviated as CK) with self-designed hydrophobic and hydrophilic amino acids arranged alternately as a template to synthesize a red / blue fluorescent reversible gold nanocluster (abbreviated as CK-AuNCs), which can present different fluorescent colors by inducing the change of the polypeptide conformation on the surface of CK-AuNCs through different solutions.

[0031] As a preferred embodiment of the present application, the specific operation of preparing the CK solution with a concentration of 2 mM is as follows: 1.5 mg of CK is dissolved in 443 µL of distilled water to obtain a CK solution with a concentration of 2 mM.

[0032] As a preferred embodiment of the present application, the preparation method of the gold nanocluster comprises the following steps:

[0033] In the CK solution with a concentration of 2 mM and a volume of 443 µL after being fully dissolved, 221 µL of a chloroauric acid solution with a concentration of 4 mM is added, followed by the addition of 14.7 µL of a 2 M NaOH solution, so as to adjust the pH value of the solution to 13. The solution is immediately mixed, wrapped with tin paper, and then transferred to a shaking incubator for reaction for 10 h to obtain H-AuNCs with bright red fluorescence.

[0034] In the embodiment of the present application, 60 µM of H-AuNCs is co-incubated with HUVEC cells for 2 h, and the cells present red fluorescence in the cells to realize bioimaging.

[0035] As a preferred embodiment of the present application, the temperature of the shaking incubator is 37℃, and the rotation speed is 180 rpm.

[0036] As a preferred embodiment of the present application, the specific operation of regulating the fluorescence of the H-AuNCs is as follows:

[0037] 200 µL of H-AuNCs is dried by using a vacuum freeze dryer for 12 h, and the freeze-dried H-AuNCs powder is taken in 200 µL of DMSO and placed in a shaking incubator to prepare D-AuNCs with bright blue fluorescence. The fluorescence of the D-AuNCs is measured after being taken out every day, so as to monitor the process of the conversion of H-AuNCs into D-AuNCs within 7 days.

[0038] In the embodiment of the present application, dimethyl sulfoxide (DMSO) organic solvent is introduced to convert into organic-phase AuNCs (D-AuNCs). See Figure 1It can be seen that the process of conversion from H-AuNCs to D-AuNCs within 7 days, the fluorescence changes from bright red to bright blue within 7 days, and the fluorescence change is linear with time.

[0039] As a preferred embodiment of the present application, the D-AuNCs are freeze-dried in a freeze dryer to obtain solid-state D-AuNCs, which are introduced into an aqueous solution, and the fluorescence changes from blue to red; the D-AuNCs solution is taken, and an aqueous solution is added, and the fluorescence changes from blue to red.

[0040] In the embodiment of the present application, the reverse regulation of D-AuNCs fluorescence and its relationship with water content are explored. 200 µL D-AuNCs are freeze-dried in a freeze dryer for 24 h to obtain solid-state D-AuNCs, which are introduced into an aqueous solution, and the fluorescence changes from blue to red within 5 s. 200 µL D-AuNCs solution is taken, and 200 µL aqueous solution is added, and the fluorescence changes from blue to red within 2 min. 140 µL D-AuNCs solution is taken, and 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50% of water solution is added, respectively, and after mixing, it is placed in a shaking incubator for 5 h, and then the fluorescence change is detected to determine the relationship between D-AuNCs solution and water content. See Figure 2 , the solvent responsiveness of D-AuNCs changes in a linear relationship within a certain range.

[0041] Exploration of the solvent color change effect of D-AuNCs

[0042] 100 µL of acetone, n-butylamine, diethyl ether, ethyl acetate, acetic acid, and ethylene glycol are selected as six types of organic solvents, which are uniformly mixed with 100 µL of D-AuNCs solution, and placed in a shaking incubator for 5 h, and then the fluorescence change is detected. In addition, 100 µL of isopropyl alcohol, ethanol, methanol, and ethylene glycol are taken, which are uniformly mixed with 100 µL of D-AuNCs solution, and placed in a shaking incubator for 5 h, and then the fluorescence change is detected to determine the relationship between D-AuNCs fluorescence and the surrounding environment and the polarity of the solvent.

[0043] See Figure 3 , acetone, n-butylamine, diethyl ether, ethyl acetate, acetic acid, and ethylene glycol all make the fluorescence of D-AuNCs change from blue to red, and this solvent color change effect is attributed to the change of the external environment of D-AuNCs, which causes the energy level splitting and rearrangement of the electrons on the surface of D-AuNCs, thereby changing the fluorescence. See Figure 4 , it can be seen that the polarity of the same type of organic solvent is positively correlated with the fluorescence conversion ability.

[0044] Research on reversible dual-fluorescence conversion mechanism

[0045] 1. Fluorescence titration: Take 200 μL D-AuNCs solution, add 0%, 10%, 20%, 30%, 40%, 50%, 60% water solution respectively, mix well, and immediately use the fluorescence spectrophotometer to detect the change of Tyr fluorescence intensity at 280 nm excitation.

[0046] Add water solution to D-AuNCs and monitor the fluorescence change of Tyr. See Figure 5 With the addition of water, the fluorescence intensity of Tyr decreases and blue shifts, which is due to the change of CK conformation in different solvents, i.e. the transition of tyrosine from surface state to encapsulated hydrophobic state. The red fluorescence of AuNCs in water solution is due to the electron donation of NH2 to the gold core, which belongs to ligand-to-gold core electron transition; the blue fluorescence of AuNCs in DMSO is due to the π-π conjugation of aromatic amino acids, which belongs to inter-ligand electron transition.

[0047] 2. NH2 modification: Take 200 μL D-AuNCs solution, add 10 μL benzaldehyde and 200 μL acetic anhydride respectively, mix well, and place in a shaking incubator. After 5 h, take out and detect the particle size to determine whether the modification is successful. Take 100 μL modified D-AuNCs, add 100 μL water, mix well, and place in a shaking incubator for 5 h. Use the fluorescence spectrophotometer to measure the fluorescence change.

[0048] See Figure 6 , the arrangement of amino acid side chains is regulated; the modification of D-AuNCs by benzaldehyde changes the hydrophilic NH2 to hydrophobic benzene ring, and the side chain changes from embedded state to surface state. The arrangement of polypeptide between hydrophilic and hydrophobic phases and between aromatic rings is adjusted, the distribution of hydrophilic and hydrophobic side chains and benzene rings on the polypeptide is changed, the number of NH2 is reduced and the number of benzene rings is increased, and the π-π conjugation of benzene rings is increased, resulting in a doubling of fluorescence intensity. The interaction between amino acids and between amino acids and gold core is analyzed; the polypeptide dissolved in DMSO has blue fluorescence, which is generated by the interaction of benzene ring side chains of polypeptide amino acids. The modification of NH2 of D-AuNCs by acetic anhydride does not produce a change from blue to red fluorescence in the water solution of acetic anhydride and the above-mentioned benzaldehyde modified D-AuNCs. This is due to the fact that NH2 cannot donate electrons to the gold core after being shielded, which destroys the interaction between amino acids and gold core, and thus cannot produce a change in fluorescence color.

[0049] The preparation method of the reversibly regulated fluorescent gold nanoclusters.

[0050] The above are only preferred embodiments of the present application, it should be pointed out that, for those skilled in the art, without departing from the concept of the present application, can also be made several variations and improvements, these should also be considered as the protection scope of the present application, these will not affect the effect and the practicality of the patent of the present application.

Claims

1. Application of reversibly regulated fluorescent gold nanoclusters in bioimaging, characterized in that, The reversible regulation fluorescent gold nanoclusters are prepared by the following steps: The H-AuNCs with bright red fluorescence are synthesized by reacting polypeptide as a template with chloroauric acid in an aqueous solution under alkaline conditions, and biological imaging is realized; the sequence of the polypeptide is CCYGGYRFKYRFK; The fluorescence of the H-AuNCs is regulated, and the specific operation is as follows: The 200 μL H-AuNCs are dried by using a vacuum freeze dryer for 12 h, the freeze-dried H-AuNCs powder is taken in 200 μL DMSO, and the D-AuNCs with bright blue fluorescence are prepared by placing in a shaking incubator; the fluorescence is measured after being taken out at intervals of 1 day, and the process of converting the H-AuNCs into D-AuNCs within 7 days is monitored.

2. Use according to claim 1, characterized in that, The D-AuNCs solution is freeze-dried in the freeze dryer to obtain the D-AuNCs in a solid state, and the fluorescence is quickly changed from blue to red after being introduced into an aqueous solution; or the D-AuNCs solution is taken, and the fluorescence is changed from blue to red after being added into an aqueous solution.

3. Use according to claim 1, characterized in that, The specific operation of preparing the polypeptide solution with a concentration of 2 mM is as follows: 1.5 mg of polypeptide is dissolved in 443 μL of distilled water to obtain a polypeptide solution with a concentration of 2 mM.

4. Use according to claim 1, characterized in that, The specific preparation steps of the reversible regulation fluorescent gold nanoclusters are as follows: The 221 μL of 4 mM chloroauric acid solution is added into the 443 μL of polypeptide solution with a concentration of 2 mM after being fully dissolved, then 14.7 μL of 2 M NaOH is added, the pH value of the solution is adjusted to 13, the solution is immediately mixed, wrapped with tin paper, and then transferred to a shaking incubator for reaction for 10 h to obtain the H-AuNCs with bright red fluorescence.

5. Use according to claim 4, characterized in that, The temperature of the shaking incubator is 37℃, and the rotation speed is 180 rpm.

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