A Bio-Thiol Responsive AIE Fluorescent Probe and Its Preparation Method and Application

Abstract

The present invention relates to a bio-thiol responsive AIE fluorescent probe and its preparation method and application. Based on the competitive regulation of AuNCs in ZIF-8, the present invention develops a unique "turn-on-turn-off-turn-on" method for highly sensitive and rapid sensing of bio-thiols. Different from traditional AIE, the aggregation-induced emission is regulated and quenched by the pre-embedded competitive factor of Hg<supgt;2+< / supgt; for the development of visual sensing nanoprobes. In the presence of bio-thiols, the mediator of Hg<supgt;2+< / supgt; competes with AuNCs based on the formation of stronger Hg<supgt;2+< / supgt>-S bonds, resulting in the recovery and enhancement of fluorescence. The designed nanoprobe can successfully respond to trace bio-thiols in serum samples. Importantly, compared with free AuNCs, its solid stability retains great potential for the sensing of bio-thiols, which can highly promote the progress of disease diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and particularly to a bio-thiol responsive AIE fluorescent probe, a preparation method thereof and an application thereof. Background Art

[0002] As important signaling molecules, bio-thiols such as cysteine (Cys) and glutathione (GSH) play important roles in physiological activities. Some studies have confirmed that abnormal levels of bio-thiols are highly correlated with neurotoxicity and cardiovascular diseases. High levels of homocysteine are reliable risk factors for hypertension, stroke and heart attack. A significant decrease in the level of GSH in serum can be used as a diagnostic marker for early Parkinson's disease. At the same time, the level of GSH is related to neurodegenerative diseases, diabetes, HIV infection and cancer. A sudden change in the molecular level of Cys may be related to liver damage, skin diseases and Alzheimer's disease. Therefore, point-of-care testing (POCT) techniques for assessing bio-thiol levels, especially visualization methods, are very necessary and have attracted more attention in analytical science.

[0003] As a class of porous crystalline materials, zeolitic imidazolate frameworks (ZIF-MOFs) have been used for adsorbing and separating various ions, or as carriers to co-encapsulate molecular probes such as enzymes, carbon dots and gold nanomaterials. The obvious improvement in the emission efficiency of AuNCs and the protective effect of the ZIF-MOFs shell pave the way for the further application of AuNCs in hazard, specific molecule sensing and sensitive POCT assessment.

[0004] In the past decade, nanosensors as an alternative method have become a promising strategy for rapidly sensing bio-thiols. Among them, gold nanoclusters (AuNCs) have been labeled as important probes for fluorescence output due to their simple synthesis method, good photostability, rapid sensing, chemical stability and biocompatibility. Nevertheless, so far, the synthesis techniques of most AuNCs always show lower fluorescence quantum yields compared with common fluorescent dyes such as rhodamine.

[0005] The regulation of the fluorescence performance of AuNCs and the improvement of the quantum yield are the application basis for later applications in the fields of sensing, imaging, etc.; currently, the synthesis techniques of most AuNCs always show lower fluorescence quantum yields. Therefore, it is of great significance to prepare a bio-thiol probe based on AIE fluorescence response. Summary of the Invention

[0006] To solve the above problems, the present invention provides a bio-thiol responsive AIE fluorescent probe, a preparation method thereof, and an application thereof. The AIE fluorescent probe of the present invention is based on the competitive regulation of AuNCs in ZIF-8, and a unique "turn-on - turn-off - enhanced turn-on" method is developed for highly sensitive and rapid sensing of bio-thiols. The synthesis of this fluorescent probe is simple and easy to use, capable of achieving highly sensitive detection of bio-thiols in solution, and reflecting the specificity and selectivity of the probe.

[0007] The present invention is achieved by the following technical solutions:

[0008] The first object of the present invention is to provide a preparation method of a bio-thiol responsive AIE fluorescent probe, comprising the following steps:

[0009] Mix a zinc source and 2-methylimidazole with an AuNCs solution respectively to obtain solution A and solution B, and mix solution A and solution B for reaction to obtain the bio-thiol responsive AIE fluorescent probe.

[0010] In an embodiment of the present invention, the AuNCs solution is prepared by the following method:

[0011] In a reaction solution, mix a solution of chloroauric acid trihydrate with glutathione and heat to obtain the AuNCs solution.

[0012] In an embodiment of the present invention, the molar ratio of chloroauric acid to glutathione is 1:1 to 1.5.

[0013] In an embodiment of the present invention, the heating conditions are: temperature 25°C to 70°C; time 20 h to 24 h.

[0014] In an embodiment of the present invention, the zinc source is selected from zinc nitrate hexahydrate and / or zinc acetate.

[0015] In an embodiment of the present invention, the molar ratio of the zinc source to 2-methylimidazole is 0.08:2 to 4.

[0016] The second object of the present invention is to provide the bio-thiol responsive AIE fluorescent probe obtained by the above preparation method.

[0017] The third object of the present invention is to provide the application of the bio-thiol responsive AIE fluorescent probe in detecting bio-thiols.

[0018] In an embodiment of the present invention, the test method of the application comprises the following steps:

[0019] (1) Mix the bio-thiol responsive AIE fluorescent probe with a quencher and incubate to obtain an AuNCs@ZIF-8 solution containing the quencher;

[0020] (2) Mix the biothiol with the AuNCs@ZIF-8 solution containing the quencher obtained in step (1), and detect the fluorescence intensity of the obtained mixture to achieve the quantitative or qualitative detection of the biothiol.

[0021] In one embodiment of the present invention, the quencher is Hg 2+ .

[0022] In one embodiment of the present invention, the biothiol is cysteine and / or glutathione.

[0023] The preparation method of the AIE fluorescent probe responsive to the biothiol of the present invention includes the following steps:

[0024] (1) Slowly drop the glutathione solution into a solution of chloroauric acid trihydrate with a certain concentration. After the two are fully mixed, continue to stir for 24 h to obtain an AuNCs solution. After the reaction, concentrate the AuNCs solution and store it at 4 °C for later use.

[0025] (2) Dissolve zinc nitrate hexahydrate and 2-methylimidazole in the AuNCs solution respectively to prepare solutions A and B. Then, under magnetic stirring at 25 °C, slowly drop solution A into solution B. After reacting for 30 min, let it stand for 2 h, and wash to obtain the AuNCs@ZIF-8 fluorescent probe.

[0026] Among them, the concentration of the chloroauric acid trihydrate in water is 20 mM; the concentration of the zinc nitrate hexahydrate solution is 0.08 M; the concentration of the 2-methylimidazole solution is 2 M to 4 M; the concentration factor of the AuNCs solution is 1.5 times.

[0027] In one embodiment of the present invention, the solutions are all water-based systems.

[0028] In one embodiment of the present invention, the full mixing is carried out by an ultrasonic-assisted dissolution method.

[0029] In one embodiment of the present invention, the concentration is carried out by vacuum drying. Specifically, place the AuNCs sample in a vacuum drying oven at 60 °C for 12 h - 24 h. After drying, redissolve the AuNCs, and then store it in the dark at 4 °C; the solvent for redissolving is water.

[0030] Preferably, the reaction route is as follows:

[0031] The application of the AIE fluorescent probe responsive to the biothiol of the present invention in the responsive detection of cysteine / glutathione and the detection of cysteine / glutathione in serum.

[0032] Preferably, the process of the responsive detection is:

[0033] (1) Take the AuNCs@ZIF-8 fluorescence sensor and measure the fluorescence intensity of the AuNCs@ZIF-8 fluorescence sensor;

[0034] (2) Add 16 μL of a solution with a concentration of 3×10 -4 M of Hg 2+ solution to the AuNCs@ZIF-8 fluorescence probe solution. After stirring and incubation, perform fluorescence emission spectrum measurement at a fixed excitation wavelength of 400 nm.

[0035] (3) Add the analyte biothiols (cysteine, glutathione solution) to the AuNCs@ZIF-8 fluorescence sensor solution containing Hg 2+ . After stirring and incubation, perform fluorescence emission spectrum measurement at a fixed excitation wavelength of 400 nm.

[0036] (4) Establish the linear relationship between the standard system and the fluorescence intensity, and calculate the contents of cysteine and glutathione in the actual sample serum.

[0037] Mechanism of the present invention:

[0038] Based on the competitive regulation of AuNCs in ZIF-8, the present invention develops a unique "turn-on - turn-off - enhanced turn-on" method for highly sensitive and rapid sensing of biothiols. Due to the self-aggregation of AuNCs, strong aggregation-induced emission is achieved. Different from traditional AIE, the aggregated luminescence is regulated and quenched by the pre-embedded competitive factor of Hg 2+ for the development of visual sensing nanoprobes. In the presence of biothiols, the mediator of Hg 2+ competes with AuNCs based on the formation of stronger Hg 2+ -S bonds, resulting in the recovery and enhancement of fluorescence. The designed nanoprobes can successfully respond to trace biothiols in serum samples. Importantly, compared with free AuNCs, its solid stability retains great potential for sensing biothiols and can highly promote the progress of disease diagnosis.

[0039] The above technical solutions of the present invention have the following advantages compared with the prior art:

[0040] (1) The synthesis of the AIE fluorescence probe described in the present invention is simple and easy to use, capable of achieving highly sensitive detection of biothiols in solution, and reflecting the specificity and selectivity of the probe.

[0041] (2) In the AIE fluorescence probe described in the present invention, AuNCs are encapsulated in ZIF-8. Due to the self-aggregation of AuNCs, strong aggregation-induced emission is achieved.

[0042] (3) The present invention regulates and quenches the aggregation-induced luminescence through the pre-embedded competitive factor of Hg 2+ . In the presence of biothiols, the mediator of Hg 2+ competes with AuNCs to form stronger Hg 2+ -S bonds, resulting in the recovery and enhancement of fluorescence.

[0043] (4) The present invention can be used to develop visual sensing probes. Based on the competitive regulation of AuNCs in ZIF-8, a unique "turn-on - turn-off - turn-on enhanced" method is developed for highly sensitive and rapid sensing of biothiols. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to make the content of the present invention easier to be clearly understood, the present invention will be further described in detail below according to the specific embodiments of the present invention in conjunction with the drawings.

[0045] Figure 1 is the high-resolution transmission electron microscope photograph of AuNCs@ZIF-8 in Example 1 of the present invention;

[0046] Figure 2 is the X-ray diffraction pattern of ZIF-8 and AuNCs@ZIF in Example 1 of the present invention;

[0047] Figure 3 is the linear fitting of the fluorescence sensing of cysteine by AuNCs@ZIF / Hg 2+ in Example 4 of the present invention;

[0048] Figure 4 is the linear fitting of the fluorescence sensing of glutathione by AuNCs@ZIF / Hg 2+ in Example 4 of the present invention;

[0049] Figure 5 is the fluorescence emission spectrum of the fluorescence probe AuNCs solution and AuNCs@ZIF-8 in Example 5 of the present invention;

[0050] Figure 6 is the fluorescence emission spectrum of the fluorescence probe AuNCs@ZIF-8 and AuNCs@ZIF-8 after co-incubation with Hg 2+ and Cys respectively in Example 5 of the present invention;

[0051] Figure 7 is the change in fluorescence intensity of the AuNCs@ZIF-8 fluorescence probe for different amino acids in Example 6 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0052] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the cited embodiments are not intended to limit the present invention.

[0053] Example 1

[0054] (1) An aqueous solution of chloroauric acid trihydrate (20 mM, 1 mL) was mixed with 8.7 mL of ultrapure water. Then, the glutathione solution (100 mM, 0.3 mL) was slowly dropped into the chloroauric acid trihydrate solution at 25 °C. After magnetic stirring at 500 rpm for 5 min, the temperature was raised to 70 °C and stirring was continued for 24 h, and it was concentrated 1.5 times to obtain an AuNCs solution, which was stored at 4 °C for later use. Concentration was carried out using vacuum drying. Specifically, the AuNCs sample was placed in a vacuum drying oven at 60 °C for drying, and the dried AuNCs was redissolved, and then stored in the dark at 4 °C; the solvent for redissolution was water.

[0055] (2) Zinc nitrate hexahydrate (0.08 M) and 2-methylimidazole (2 M) were respectively dissolved in the concentrated aqueous solution to prepare solutions A and B. Then, solution A was slowly dropped into solution B under magnetic stirring (500 rpm) at 25 °C. After reacting for 30 min, it was left standing for 2 h and centrifuged 3 times (10000 rpm, 10 min) to obtain the ZIF-8 probe.

[0056] (3) Zinc nitrate hexahydrate (0.08 M) and 2-methylimidazole (2 M) were respectively dissolved in the concentrated AuNCs solution to prepare solutions A and B. Then, solution A was slowly dropped into solution B under magnetic stirring (500 rpm) at 25 °C. After reacting for 30 min, it was left standing for 2 h and centrifuged 3 times (10000 rpm, 10 min) to obtain the AuNCs@ZIF-8 fluorescent probe.

[0057] Figure 1 This is the high-resolution transmission electron micrograph of ZIF-8 and AuNCs@ZIF-8 nanoparticles in this example. From Figure 1 It can be seen that ZIF-8 has a regular and uniform polyhedral shape with a uniform size distribution of about 120 nm. The one-pot synthesized AuNCs@ZIF-8 particles only show a slight increase in shape and size. In addition, the AuNCs@ZIF-8 particle image shows that the AuNCs particles are encapsulated within the ZIF-8 framework rather than adsorbed on the surface, which provides evidence for aggregation-induced emission.

[0058] Figure 2X-ray diffraction patterns of ZIF-8 and AuNCs@ZIF-8 in Example 1. From the combined X-ray diffraction patterns of ZIF-8 and AuNCs@ZIF-8, it can be seen that the X-ray diffraction pattern of AuNCs@ZIF-8 is almost the same as that of ZIF-8, indicating that the ZIF-8 framework in AuNCs@ZIF-8 is not destroyed by the internal AuNCs particles.

[0059] Example 2

[0060] (1) An aqueous solution of chloroauric acid trihydrate (20 mM, 1 mL) was mixed with 8.7 mL of ultrapure water. Then, the glutathione solution (100 mM, 0.3 mL) was slowly added dropwise to the chloroauric acid trihydrate solution at 25 °C. After magnetic stirring at 500 rpm for 5 min, the temperature was raised to 70 °C and stirring continued for 24 h. The solution was concentrated 1.5-fold to obtain an AuNCs solution, which was stored at 4 °C for later use.

[0061] (2) Zinc nitrate hexahydrate (0.08 M) and 2-methylimidazole (4 M) were separately dissolved in the concentrated AuNCs solution to prepare solutions A and B. Then, solution A was added dropwise to solution B under magnetic stirring (500 rpm) at 25 °C. After reacting for 30 min, the mixture was left standing for 2 h and centrifuged three times (10,000 rpm, 10 min) to obtain the AuNCs@ZIF-8 fluorescent probe.

[0062] Example 3

[0063] (1) An aqueous solution of chloroauric acid trihydrate (20 mM, 1 mL) was mixed with 8.7 mL of ultrapure water. Then, the glutathione solution (100 mM, 0.1 mL) was slowly added dropwise to the chloroauric acid trihydrate solution at 25 °C. After magnetic stirring at 500 rpm for 5 min, the temperature was raised to 70 °C and stirring continued for 24 h. The solution was concentrated 1.5-fold to obtain an AuNCs solution, which was stored at 4 °C for later use.

[0064] (2) Zinc nitrate hexahydrate (0.08 M) and 2-methylimidazole (4 M) were separately dissolved in the concentrated AuNCs solution to prepare solutions A and B. Then, solution A was added dropwise to solution B under magnetic stirring (500 rpm) at 25 °C. After reacting for 30 min, the mixture was left standing for 2 h and centrifuged three times (10,000 rpm, 10 min) to obtain the AuNCs@ZIF-8 fluorescent probe.

[0065] Responsiveness detection of biothiols in Example 4

[0066] (1) Take the AuNCs@ZIF-8 fluorescent probe and measure the fluorescence intensity of the AuNCs@ZIF-8 fluorescence sensor;

[0067] (2) Add 16 μL of a solution with a concentration of 3×10 -4 M of Hg 2+ solution to the AuNCs@ZIF-8 fluorescent probe solution. After stirring and incubation, an AuNCs@ZIF-8 solution containing Hg 2+ (AuNCs@ZIF-8 / Hg 2+ solution) is obtained. With a fixed excitation wavelength of 400 nm, fluorescence emission spectra are measured.

[0068] (3) Add different concentrations of Cys solutions (0.0 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 4.0 μM, 5.0 μM, 8.0 μM, 10.0 μM, 20.0 μM, and 30.0 μM) to 5 μL of the AuNCs@ZIF-8 solution containing Hg 2+ respectively. After shaking well, AuNCs@ZIF-8 / Hg 2+ / Cys solutions are obtained. Take 1 mL of each AuNCs@ZIF-8 / Hg 2+ / Cys solution and detect the fluorescence intensity with a fluorescence spectrometer. Parameter settings: excitation wavelength is 500 nm - 750 nm, scanning speed is 1 nm / min, and excitation and emission slits are 10 nm. The detection results are as Figure 3 shown. It can be seen from Figure 3 that there is a linear relationship between the content of Cys and the fluorescence intensity.

[0069] Add different concentrations of GSH solutions (0.0 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 4.0 μM, 5.0 μM, 8.0 μM, 10.0 μM, 20.0 μM, and 30.0 μM) to 5 μL of the AuNCs@ZIF-8 (3×10 2+ M, 8 μL) solution containing Hg -4 respectively. After shaking well, AuNCs@ZIF-8 / Hg 2+ / Cys solutions are obtained. Take 1 mL of each AuNCs@ZIF-8 / Hg 2+ / Cys solution and detect the fluorescence intensity with a fluorescence spectrometer. Parameter settings: excitation wavelength is 500 nm - 750 nm, scanning speed is 1 nm / min, and excitation and emission slits are 10 nm. The detection results are as Figure 4 shown. It can be concluded from Figure 4 that there is a linear relationship between the content of glutathione and the fluorescence intensity.

[0070] Example 5 Fluorescence Feasibility Analysis

[0071] The fluorescence detection feasibility analysis of the AuNCs solution and AuNCs@ZIF-8 prepared in Example 1 was carried out using a fluorescence spectrometer, and the results are as follows Figure 5 shown. It can be seen from Figure 5 that the fluorescence intensity of AuNCs@ZIF-8 is greatly enhanced compared with that of the AuNCs solution with the same molar concentration, about 6 times.

[0072] Previous studies have shown that the enhanced luminescence of AuNCs is related to the reduction of non-radiative decay caused by the restricted intramolecular motion. Therefore, the limited space in ZIF-8 prevents intramolecular rotation, providing an unstable aggregation for AuNCs, ultimately leading to the aggregation-induced emission (AIE) phenomenon. In this invention, the fluorescence detection feasibility analysis of the AuNCs@ZIF-8 solution, AuNCs@ZIF-8 / Hg 2+ solution, and AuNCs@ZIF-8 / Hg 2+ / Cys solution obtained in Example 1 and Example 4 was carried out, and the results are as follows Figure 6 shown. It is shown that when Hg Figure 6 is added to the fluorescence probe solution, the fluorescence intensity will rapidly decrease. This may be due to the formation of a metal bond between Hg 2+ and Au 2+ on the surface of AuNCs. On the other hand, when Cys molecules are added, the quenched fluorescence will gradually recover. This quenched and recovered fluorescence may be due to the reaction between Hg + ions and sulfhydryl groups being stronger and more stable than the reaction between AuNC and Hg 2+ ions. 2+

[0073] Fluorescence selectivity analysis of Example 6

[0074] Solutions of different amino acids (Asp, Arg, Lys, Gly, Val, Ile, Leu, His, Phe, Thr, Pro, Ala, Glu, Trp, Ser, Met, Gln, Asn, Tyr) (concentration 10 times that of the Cys solution) were respectively prepared, and the prepared amino acid solutions were respectively added to 5 μL of the AuNCs@ZIF-8 (3×10 2+ M, 8 μL) solution containing Hg -4 . After shaking well, 1 mL of the amino acid solution was taken respectively. Finally, the fluorescence spectrum was measured using a fluorescence spectrometer.

[0075] In the presence of other amino acids (Asp, Arg, Lys, Gly, Val, Ile, Leu, His, Phe, Thr, Pro, Ala, Glu, Trp, Ser, Met, Gln, Asn, and Tyr), the fluorescence sensing performance of the fluorescent probe AuNCs@ZIF-8 for biothiols (GSH, Cys solutions) is as Figure 7 shown. As Figure 7 can be seen, even in the presence of other common amino acids, the AuNCs@ZIF-8 of the present invention still has good selectivity for the fluorescence detection of biothiols (GSH, Cys solutions), indicating that it has good anti-interference ability and can be used for the fluorescence sensing of biothiols (GSH, Cys solutions).

[0076] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. The obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. Application of biothiol-responsive AIE fluorescent probe in detecting biothiols, characterized in that, the method of the application includes the following steps: (1) Mix and incubate the biothiol-responsive AIE fluorescent probe with a quencher to obtain an AuNCs@ZIF-8 solution containing the quencher; (2) Mix the biothiol with the AuNCs@ZIF-8 solution containing the quencher obtained in step (1), and detect the fluorescence intensity of the obtained mixture to achieve quantitative or qualitative detection of the biothiol; The quenching agent is Hg 2+ ; The preparation method of the biothiol-responsive AIE fluorescent probe includes the following steps: Mix the zinc source and 2-methylimidazole with the AuNCs solution respectively to obtain solution A and solution B, and mix and react solution A and solution B to obtain the biothiol-responsive AIE fluorescent probe; The AuNCs solution is prepared by the following method: In the reaction solution, mix and heat chloroauric acid and glutathione to obtain the AuNCs solution.

2. The application according to claim 1, characterized in that, the molar ratio of chloroauric acid to glutathione is 1:1~1.

5.

3. The application according to claim 1, characterized in that, the zinc source is selected from zinc nitrate hexahydrate and / or zinc acetate.

4. The application according to claim 1, characterized in that, the molar ratio of the zinc source to 2-methylimidazole is 0.08:2~4.

5. The application according to claim 1, characterized in that, the biothiol is cysteine and / or glutathione.

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