A carbon dot for detecting and inhibiting protein aggregation, and preparation and application thereof

Abstract

The present invention relates to carbon dots for detecting and inhibiting protein aggregation, and their preparation and application, belonging to the technical field of biomedicine. The carbon dots of the present invention are prepared by a one-pot method from a reducing carbon source, congo red, and an oxidant containing a peroxide bond. The carbon dots prepared by the present invention have multiple functions of red fluorescence turn-on imaging of protein aggregates, protein aggregation inhibition, and reactive oxygen species scavenging, and can be used for the applications of detecting protein aggregates, inhibiting protein aggregation, and scavenging reactive oxygen species. The preparation method of the multifunctional carbon dots provided by the present invention is simple, easy to implement, and low in cost, suitable for large-scale production. The red fluorescence response signal of the carbon dots has strong anti-photobleaching ability, and has great application prospects in protein aggregation diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to carbon dots for detecting and inhibiting protein aggregation, and their preparation and application. In particular, it relates to a class of carbon dots with both detection and treatment functions for protein aggregation, and specifically to a preparation method and application of a class of multifunctional carbon dots with protein aggregate fluorescence imaging, protein aggregation inhibition, and reactive oxygen species scavenging functions. Background Art

[0002] Proteins are an important material basis for the composition of organisms, participating in various links in the body's biochemical reaction network, and are the main bearers of life activities. The structure of proteins is crucial for maintaining their normal physiological functions. However, under pathological conditions, some proteins undergo misfolding and aggregation, transforming from soluble protein monomers into insoluble protein aggregates rich in β-sheet structures, which are deposited in specific tissues or organs. This pathological phenomenon not only causes the corresponding proteins to lose their normal physiological activities, but the generated intermediate aggregates (such as oligomers and protofibrils) may also cause cytotoxic effects such as cell membrane damage, organelle dysfunction, and cellular oxidative stress, and have been proven to be closely related to the occurrence and development of various neurodegenerative diseases and metabolic diseases. For example, Alzheimer's disease (AD) associated with the aggregation of β-amyloid protein (Aβ), Parkinson's disease (PD) associated with the aggregation of α-synuclein (α-syn), and type II diabetes (T2DM) associated with the aggregation of human islet amyloid polypeptide (IAPP) and insulin, etc.

[0003] Regarding the pathological changes of protein aggregation, on the one hand, as important pathological markers of related diseases, such as senile plaques in the brains of AD patients and Lewy bodies in the brains of PD patients, the development of effective imaging detection methods is of great significance for the early diagnosis of diseases. On the other hand, in order to avoid the cytotoxicity caused by protein aggregation and subsequent organ dysfunction, inhibiting protein aggregation is considered an important treatment strategy. In addition, since the development of this pathological process is inseparable from the generation of excessive reactive oxygen species, therefore, scavenging reactive oxygen species while inhibiting aggregation and reducing the level of oxidative stress helps to further reduce the corresponding pathological damage. More importantly, the development of probes with the potential for integrated diagnosis and treatment applications, by combining detection and treatment functions, is expected to achieve real-time feedback of treatment effects with detection signals and guide the treatment process, with the advantages of being safer and more efficient. However, the development of such probes still faces great challenges.

[0004] Fluorescence imaging is a method with low invasiveness and can achieve in-vivo detection, having broad application prospects in the field of disease diagnosis. As a carbon-based nanomaterial with excellent fluorescence properties, carbon dots also have advantages such as good biocompatibility, low toxicity, and low preparation cost, and are widely used in the biomedical field, including the detection of protein aggregation disease markers, as well as therapeutic research such as inhibiting protein aggregation or dissociating protein aggregates. However, the fluorescence response signals of most carbon dots are blue-green light, and the detection process is easily interfered by the spontaneous blue fluorescence of biological samples. Moreover, carbon dots with both detection and therapeutic dual application capabilities are still very limited, and their preparation methods still need to be further expanded. Therefore, developing carbon dots with red fluorescence detection performance for protein aggregates and having a therapeutic intervention effect on the protein aggregation process is of great significance for the early diagnosis and treatment research of related diseases, and helps to further expand the application of carbon dots in the field of disease diagnosis and treatment. Summary of the Invention

[0005] The object of the present invention is to overcome the limitations of carbon dots in the diagnosis and treatment of protein aggregation diseases, and provide a preparation method of multifunctional carbon dots with certain general applicability, which can be used to prepare a class of multifunctional carbon dots that simultaneously have red fluorescence turn-on imaging of protein aggregates, protein aggregation inhibition, and reactive oxygen species scavenging. The preparation method of this class of carbon dots is simple and easy to operate, with low cost. The prepared carbon dots have both detection and therapeutic dual application functions, and can be further used for the preparation of diagnostic reagents, therapeutic drugs, or integrated diagnosis and treatment drugs for protein aggregation diseases.

[0006] According to the first aspect of the present invention, there is provided a preparation method of carbon dots for detecting and inhibiting protein aggregation, comprising the following steps:

[0007] (1) Dissolve a carbon source, congo red, and an oxidant in deionized water, and heat for reaction; then centrifuge to collect the supernatant; the carbon source has reducibility, and the oxidant contains a peroxy bond;

[0008] (2) Dialyze the supernatant obtained in step (1), take out the inner dialysis solution after dialysis, and heat to obtain a carbon dot concentrate.

[0009] Preferably, the carbon source is at least one of citric acid, ethylenediamine, and ascorbic acid.

[0010] Preferably, the oxidant is at least one of ammonium persulfate, benzoyl peroxide, and hydrogen peroxide.

[0011] Preferably, the concentration of the carbon source in the deionized water is 0.2 - 3 mol / L, the concentration of the oxidant is 0.002 - 0.5 mol / L, and the concentration of congo red is 0.2 - 20 g / L.

[0012] Preferably, in step (1), the heating temperature is 100 - 300 °C and the time is 5 min - 8 h.

[0013] On the other hand, according to the present invention, there is provided carbon dots prepared by any of the above methods.

[0014] On the other hand, according to the present invention, there is provided the application of the carbon dots in the preparation of a protein aggregate detection reagent.

[0015] On the other hand, according to the present invention, there is provided the application of the carbon dots in the preparation of a protein aggregate inhibitor drug.

[0016] Preferably, the protein aggregate is an aggregate containing a β-sheet structure formed by an aggregating protein;

[0017] Preferably, the aggregating protein is at least one of β-amyloid protein, α-synuclein, human islet amyloid polypeptide, insulin, huntingtin, superoxide dismutase, prion protein, and lysozyme.

[0018] On the other hand, according to the present invention, there is provided the application of the carbon dots in the preparation of a reactive oxygen species scavenging drug.

[0019] Generally speaking, compared with the prior art, the above technical solutions conceived by the present invention mainly have the following technical advantages:

[0020] (1) The raw materials for preparing the carbon dots provided by the present invention are cheap and easily available, the preparation method is simple and easy to implement, without a complicated post-modification process, and its time and economic costs are both low, which is suitable for large-scale production.

[0021] (2) The method for preparing the carbon dots provided by the present invention can be used to prepare a variety of carbon dots with multiple functions of red fluorescence turn-on imaging of protein aggregates, protein aggregation inhibition, and reactive oxygen species scavenging. In terms of detection, based on the response molecule congo red, and the incorporation of a reducing carbon source and an oxidant, the large conjugate structure formed between the electron-withdrawing group and the electron-donating group on the surface of the prepared carbon dots and the carbon core may endow them with red fluorescence response performance. The red fluorescence response signal of the carbon dots has the ability to avoid the interference of spontaneous blue fluorescence of biological samples, and the red fluorescence of the carbon dots has better anti-photobleaching properties than congo red, indicating that it has great application prospects in biological imaging and early disease diagnosis; in terms of treatment, this type of carbon dots not only has the function of effectively inhibiting protein aggregation, but also can scavenge reactive oxygen species to reduce oxidative stress damage.

[0022] (3) The carbon dots provided by the present invention have dual application functions of detection and treatment, have the potential for integrated diagnosis and treatment applications, and provide a new method for the research and development of diagnostic reagents, therapeutic drugs, or integrated diagnosis and treatment drugs for protein aggregation diseases. Brief Description of the Drawings

[0023] Figure 1 Transmission electron microscopy image and particle size distribution diagram of CA-CDs;

[0024] Figure 2 Transmission electron microscopy image and particle size distribution diagram of EDA-CDs;

[0025] Figure 3 Fluorescence microscope images of insulin aggregates and Aβ aggregates labeled with Congo red, CA-CDs and EDA-CDs, and fluorescence spectra of Congo red, CA-CDs and EDA-CDs for detecting insulin monomers and insulin aggregates; 42

[0026] Figure 4 Comparison of the anti-photobleaching properties of insulin aggregates labeled with Congo red, CA-CDs and EDA-CDs;

[0027] Figure 5 Results of the inhibitory effect of CA-CDs and EDA-CDs on insulin aggregation, where (A) shows the ThT fluorescence test results during the co-incubation of insulin with different concentrations of CA-CDs and EDA-CDs for 0 - 1 h, and (B) circular dichroism spectra and (C) TEM images of insulin itself and after co-incubation with CA-CDs and EDA-CDs (200 μg / mL) for 1 h.

[0028] Figure 6 Results of the inhibitory effect of CA-CDs and EDA-CDs on Aβ 42 aggregation, where (A) shows the ThT fluorescence test results during the co-incubation of different concentrations of CA-CDs with Aβ 42 for 0 - 6 h, and (B) circular dichroism spectra and (C) TEM images of Aβ 42 itself and after co-incubation with CA-CDs and EDA-CDs (200 μg / mL) for 6 h.

[0029] Figure 7 Scavenging rates of different concentrations of CA-CDs, EDA-CDs, citric acid and ascorbic acid on (A) DPPH · , (B) ·OH and (C) O2 ·- .

[0030] Figure 8 Results of the scavenging effect of CA-CDs and EDA-CDs on reactive oxygen species in SH-SY5Y cells induced by Aβ 42 , where (A) shows the relative fluorescence intensity of DCF in cells, and (B) shows the fluorescence and bright field images of cells.

[0031] Figure 9 ​The cytotoxicity of CA-CDs and EDA-CDs (A) themselves and (B) their protective effects against Aβ-induced cytotoxicity. 42 induced cytotoxicity. Detailed implementation manners

[0032] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] A preparation method of a class of carbon dots with both detection and treatment functions for protein aggregation provided by the present invention includes the following steps:

[0034] Step (1): Dissolve a carbon source with certain reducibility, congo red and an oxidant in deionized water, heat and react, after cooling to room temperature, centrifuge at 10,000 rpm for 8 min, and collect the supernatant solution;

[0035] Step (2): Transfer the supernatant solution obtained in (1) to a dialysis bag with a molecular cut-off of 1000 Da, use deionized water as the external dialysis solution for dialysis for 48 h, and regularly change the external dialysis solution during the dialysis process;

[0036] Step (3): Take out the internal dialysis solution obtained in (2), heat at 80 °C to obtain a carbon dot concentrate, quantify it, and store it in the dark.

[0037] Among them, the carbon source described in step (1) refers to a small molecule reagent with reducibility, including but not limited to citric acid, ethylenediamine and ascorbic acid, and the oxidant refers to a small molecule reagent containing a peroxy bond (O-O), including but not limited to ammonium persulfate, benzoyl peroxide and hydrogen peroxide. The concentration of the carbon source is 0.2 - 3 mol / L, the concentration of the oxidant is 0.002 - 0.5 mol / L, and the concentration of congo red is 0.2 - 20 g / L. The heating reaction methods include but are not limited to oil bath condensation reflux heating method, autoclave hydrothermal method and microwave heating method, the heating reaction temperature is 100 - 300 °C, and the reaction time is 5 min - 8 h.

[0038] The carbon dots prepared by the present invention have the detection function of red fluorescence turn-on imaging for protein aggregates rich in β-sheet structures, and at the same time have an inhibitory effect on protein aggregation, and can scavenge reactive oxygen species, having a therapeutic effect on the pathological process of protein aggregation. And this class of carbon dots combines detection and treatment functions, and has the application prospect of diagnosis and treatment integration.

[0039] The present invention provides any one of the following applications of the carbon dots with both detection and treatment functions for protein aggregation:

[0040] (1) For red fluorescence turn-on imaging of protein aggregates;

[0041] (2) For inhibiting protein aggregation;

[0042] (3) For scavenging reactive oxygen species;

[0043] (4) For preparing diagnostic reagents, therapeutic drugs or theranostic drugs for protein aggregation diseases.

[0044] Among them, the proteins described in applications (1) and (2) refer to aggregating proteins, including but not limited to Aβ, α-syn, IAPP, insulin, huntingtin, superoxide dismutase, prion protein and lysozyme, and the protein aggregates refer to aggregates rich in β-sheet structure formed by the aggregating proteins or the formed deposits. The protein aggregation diseases described in application (4) include but not limited to AD, PD, T2DM, Huntington's chorea, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease and familial visceral amyloidosis.

[0045] Example 1

[0046] Using citric acid as the carbon source and ammonium persulfate as the oxidant to prepare multifunctional carbon dots (CA-CDs), the preparation steps are as follows:

[0047] Dissolve 15 g of citric acid, 50 mg of congo red and 0.5 g of ammonium persulfate in 50 mL of deionized water, place it in a round-bottom flask, and carry out a condensation reflux stirring reaction in an oil bath at 180 °C for 4 h. After the reaction is completed, wait for the reaction product to cool naturally to room temperature, centrifuge at 10000 rpm for 8 min, and collect the supernatant. Then transfer it to a dialysis bag with a molecular cut-off of 1000 Da and dialyze it with deionized water for 48 h, and regularly change the external dialysis solution to fully remove small molecule impurities. When the pH of the external dialysis solution is neutral, it is judged that the dialysis is completed. Finally, take out the internal dialysis solution, heat and concentrate it at 80 °C and quantify it, and store it in the dark for use, which is CA-CDs. Observe the morphology of CA-CDs through a Talos F200X transmission electron microscope (TEM), as Figure 1 shown, the carbon dots are well-dispersed quasi-spherical, with an average particle size of 2.2 ± 0.6 nm and a lattice spacing of 0.20 nm.

[0048] Example 2

[0049] Using ethylenediamine as the carbon source and ammonium persulfate as the oxidant to prepare multifunctional carbon dots (EDA-CDs), the preparation steps are as follows:

[0050] Dissolve 5.22 mL of ethylenediamine, 50 mg of congo red, and 0.5 g of ammonium persulfate in 50 mL of deionized water, place it in a round-bottom flask, and carry out a condensation reflux stirring reaction in an oil bath at 180 °C for 4 h. After the reaction is completed, allow the reaction product to cool naturally to room temperature, centrifuge at 10000 rpm for 8 min, and collect the supernatant solution. Then transfer it to a dialysis bag with a molecular cut-off of 1000 Da and dialyze it with deionized water for 48 h, and regularly change the external dialysis solution to fully remove small molecule impurities. When the pH of the external dialysis solution is neutral, it is judged that the dialysis is completed. Finally, take out the internal dialysis solution, heat and concentrate it at 80 °C and quantify it, and store it in the dark for later use, which is EDA-CDs. TEM characterizes the morphology of EDA-CDs, as Figure 2 shown. The carbon dots have good dispersibility, the average particle size is 2.3 ± 0.4 nm, and the lattice spacing is 0.21 nm.

[0051] Example 3

[0052] The red fluorescence turn-on imaging detection of CA-CDs and EDA-CDs obtained in Example 1 and Example 2 for insulin and β-amyloid 1-42 (Aβ 42 ) aggregates includes the following steps:

[0053] (1) Preparation of insulin aggregates: Dissolve insulin dry powder in HCl-KCl solution (50 mM, pH 2) to form a 40 μM incubation solution, and magnetically stir it in a 65 °C water bath for 1 h to obtain the Ins-Fibril mother liquor (40 μM, the concentration is calculated based on the monomer concentration), and store it at -20 °C for later use.

[0054] Aβ 42 Preparation of aggregates: Dissolve Aβ 42 dry powder in NaOH solution (60 mM) to prepare a 200 μM mother liquor, and dilute it with 50 mM phosphate buffer solution (containing 100 mM NaCl, pH 7.4) to a 20 μM incubation solution, and statically incubate it in a 37 °C water bath for 6 h to obtain Aβ 42 -Fibril mother liquor (20 μM, the concentration is calculated based on the monomer concentration), and store it at -20 °C for later use.

[0055] (2) Fluorescent imaging of protein aggregates: Dilute the insulin aggregates or Aβ 42 aggregates prepared in step (1) to 4 μM with phosphate buffer (10 mM, pH 7.4), add 50 μg / mL congo red or CA-CDs or EDA-CDs prepared in Example 1 or 2, co-incubate for 10 min, and place it in a 96-well plate to observe the fluorescence using a fluorescence microscope.

[0056] (3) Fluorescence spectral detection of protein aggregates: Taking insulin as an example, dilute insulin monomers or the insulin aggregates prepared in step (1) to 8 μM with phosphate buffer, add 50 μg / mL congo red or the CA-CDs or EDA-CDs prepared in Example 1 or 2, incubate for 10 min, record the fluorescence emission spectrum, and the fluorescence detection conditions are excitation wavelength 510 nm and emission wavelength range 520 - 800 nm.

[0057] (4) Comparison of anti-photobleaching performance in fluorescence imaging: Taking insulin aggregates as an example, dilute the insulin aggregates prepared in step (1) to 4 μM with phosphate buffer (10 mM, pH 7.4), add 50 μg / mL congo red or the CA-CDs or EDA-CDs prepared in Example 1 or 2, incubate for 10 min, drop the co-incubation solution on a glass slide, wait for it to dry naturally, and then observe it under the red light channel of a fluorescence microscope under continuous light for 15 min and collect images.

[0058] The results of fluorescence imaging and spectral detection are as Figure 3 shown. Under the red fluorescence channel, it was observed that red fluorescence appeared after CA-CDs and EDA-CDs labeled insulin aggregates or Aβ 42 aggregates, while the two carbon dot solutions themselves did not have red fluorescence. Moreover, the fluorescence spectral detection results using insulin as a test model showed that the emission peaks in the red fluorescence region were significantly enhanced after CA-CDs and EDA-CDs were co-incubated with insulin aggregates, while no response signal was observed for insulin monomers, indicating that CA-CDs and EDA-CDs have the same red fluorescence turn-on imaging performance as congo red dye for the above two protein aggregates.

[0059] The comparison results of anti-photobleaching performance in fluorescence imaging are as Figure 4 shown. The red fluorescence produced by congo red labeling decreased significantly after continuous light irradiation for 3 min and almost disappeared after 10 min. In contrast, after CA-CDs and EDA-CDs labeling, obvious red fluorescence was still observed after continuous light irradiation for 10 min, and the insulin aggregates labeled with red fluorescence could still be observed until 15 min, indicating that CA-CDs and EDA-CDs have better anti-photobleaching properties than congo red.

[0060] Example 4

[0061] The inhibitory effects of CA-CDs and EDA-CDs obtained in Example 1 and Example 2 on insulin and Aβ 42 aggregation include the following investigation steps:

[0062] (1) Take CA-CDs or EDA-CDs at different concentrations (0, 10 μg / mL, 50 μg / mL, and 200 μg / mL) and incubate them with insulin or Aβ 42 for co-incubation. The incubation conditions are the same as those in step (1) of Example 3, except that samples need to be taken at specific time points during the incubation for thioflavin T (ThT) fluorescence detection.

[0063] (2) ThT fluorescence detection: Dilute the co-incubation samples taken in step (1) with phosphate buffer to a specific concentration (4 μM for insulin aggregates and 1 μM for Aβ 42 aggregates), add 10 μM ThT solution and mix well, and detect the fluorescence intensity of the samples (excitation wavelength is 430 nm, emission wavelength is 488 nm). Take the difference between the fluorescence intensity at different incubation times and the fluorescence intensity at 0 hour of the same sample as the absolute fluorescence intensity, and set the maximum absolute fluorescence intensity of the protein aggregates incubated without carbon dots as 100%, and normalize the data of each sample to obtain the relative fluorescence intensity.

[0064] (3) Circular dichroism spectroscopy detection: Take 400 μL of the incubation solution of insulin aggregates (4 μM) or Aβ 42 aggregates (20 μM) incubated alone or with carbon dots for 1 h or 6 h, add it to a circular dichroism cuvette with an optical path of 1 mm, scan the signal values in the wavelength range of 200 - 250 nm, the scanning speed is 200 nm / min, and the spectral bandwidth is 2 nm.

[0065] (4) TEM test: Take 10 μL of the co-incubation sample at the final time point prepared in step (1), drop it onto a copper mesh supported by an ultra-thin carbon film, let it stand for 5 min, then drop deionized water and wash three times, and finally drop freshly prepared 2% phosphotungstic acid solution for negative staining, and observe by TEM after air-drying at room temperature.

[0066] The experimental results are as Figure 5 and Figure 6 shown. After co-incubation of CA-CDs and EDA-CDs with insulin and / or Aβ 42 , the relative ThT fluorescence intensity decreased in a carbon dot concentration-dependent manner, indicating that the above two carbon dots can inhibit the aggregation of insulin and / or Aβ 42 , and then form aggregates with fewer β-sheet structures. The results of circular dichroism spectroscopy test show that after co-incubation with CA-CDs and EDA-CDs, the signal peaks of the formed insulin and Aβ 42 aggregates at 220 nm both decreased, further verifying that in the presence of the above two carbon dots, the aggregates formed by insulin and Aβ 42 have fewer β-sheet structures. And the TEM test results show that compared with insulin and Aβ 42Typical fibrous aggregates formed by self-aggregation. In the presence of CA-CDs and EDA-CDs, both of the above two proteins only formed smaller aggregate structures. In summary, ThT fluorescence test, circular dichroism spectroscopy test, and TEM test all showed that CA-CDs and EDA-CDs could effectively inhibit the aggregation of insulin and Aβ 42 .

[0067] Example 5

[0068] The scavenging activities of CA-CDs and EDA-CDs obtained in Example 1 and Example 2 against intracellular and extracellular reactive oxygen species include the following investigation steps:

[0069] (1) Scavenging of reactive oxygen species in vitro

[0070] A. Investigation of DPPH radical (DPPH · ) scavenging: Different concentrations of CA-CDs or EDA-CDs (0, 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL) were added to 120 μM DPPH · ethanol solution, shaken in the dark for 1 h, the absorbance value at 517 nm was measured, and the scavenging rate was calculated.

[0071] B. Investigation of hydroxyl radical (·OH) scavenging: First, 3.3 mM H2O2 was added to 1.2 mM FeSO4 aqueous solution, after standing for 10 min, 1.2 mM salicylic acid was added, and the solution quickly turned purple. 160 μL of the above solution was mixed with 40 μL of CA-CDs or EDA-CDs at a specific concentration, shaken in the dark for 0.5 h, the absorbance value of the mixed solution at 520 nm was measured, and the scavenging rate was calculated.

[0072] C. Investigation of superoxide anion (O2 ·- ) scavenging: First, pyrogallol was dissolved in 10 mM HCl solution to prepare a 5 mM pyrogallol solution for use. Then, 150 μL of Tris-HCl solution (50 mM, pH 8.2) was mixed with 40 μL of carbon dots at a specific concentration, 10 μL of the freshly prepared pyrogallol solution was added, shaken in the dark for 10 min, the absorbance value at 320 nm was measured, and the scavenging rate was calculated.

[0073] The results of the in vitro scavenging of reactive oxygen species (DPPH · , ·OH and O2 ·- ) by CA-CDs and EDA-CDs are as Figure 7 shown. CA-CDs showed scavenging effects on DPPH · , ·OH and O2 ·-All three types of reactive oxygen species have good scavenging effects, and the overall effect is better than that of the carbon source citric acid. EDA-CDs have good scavenging effects on DPPH · and O2 ·- but have a relatively low scavenging rate for ·OH. Generally speaking, since CA-CDs and EDA-CDs have good scavenging activities for DPPH · it indicates that CA-CDs and EDA-CDs have good antioxidant activities.

[0074] (2) Intracellular reactive oxygen species scavenging

[0075] Human neuroblastoma cells (SH-SY5Y) were selected as the cell research model. SH-SY5Y cells were seeded into black 96-well plates (10,000 cells / well) and cultured in a cell incubator (37 °C, 5% CO2) for 16 h, and then the original culture medium was discarded. Fresh medium containing 40 μM Aβ 42 and 0 or 200 μg / mL CA-CDs or EDA-CDs was added and cultured for 4 h. The blank control group was only added with the medium. After 4 h, the original culture medium was discarded, and the cells were gently washed three times with DMEM. Then, a DMEM solution containing 10 μM 2’,7’-dichlorofluorescein diacetate (DCFH-DA) was added to each well, and the cells were incubated for another 30 min. Then, the supernatant was carefully aspirated, and the cells were gently washed three times with DMEM. Finally, 100 μL of DMEM was added to each well, and the fluorescence value was measured using a microplate reader (excitation wavelength: 485 nm, emission wavelength: 528 nm), and the fluorescence of the cells in the wells was observed using an inverted fluorescence microscope.

[0076] The results of CA-CDs and EDA-CDs scavenging intracellular reactive oxygen species are as Figure 8 shown. After Aβ 42 stimulation, the DCF fluorescence intensity in SH-SY5Y cells increased by about 93%. Obvious green fluorescence was observed in the cells under the microscope, indicating that Aβ 42 stimulated the cells to produce excessive reactive oxygen species. When CA-CDs and EDA-CDs were added, the DCF fluorescence intensity of the cells decreased by 26.8% and 11.4% respectively, and the green fluorescence intensity of the cells observed under the microscope also decreased correspondingly, indicating that CA-CDs and EDA-CDs have a certain scavenging effect on Aβ 42 induced intracellular reactive oxygen species.

[0077] Example 6

[0078] The protective effects of CA-CDs and EDA-CDs obtained in Example 1 and Example 2 against Aβ 42 induced cytotoxicity were investigated as follows:

[0079] Investigation of the cytotoxicity of carbon dots: SH-SY5Y cells were seeded into 96-well plates (10,000 cells / well) and cultured in a cell incubator (37 °C, 5% CO2) for 16 h. The original culture medium was discarded, and fresh medium containing different concentrations of carbon dots (0, 1 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 600 μg / mL) was added and cultured for another 24 h. The original culture medium was discarded again, and after washing three times with DMEM, a DMEM solution containing 10% CCK-8 (v / v) was added to each well. After incubation for 1 h, the absorbance value of each well was measured (λ = 450 nm), and the cell survival rate was calculated.

[0080] The protective effect of carbon dots on Aβ 42 induced cytotoxicity was investigated: First, the Aβ 42 dry powder was dissolved in DMSO to a 4 mM solution, and then diluted to 400 μM with 1×PBS and sonicated for 5 min for later use. SH-SY5Y cells were seeded into 96-well plates (10,000 cells / well) and cultured in a cell incubator (37 °C, 5% CO2) for 16 h. The original culture medium was discarded, and different concentrations of carbon dots (10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL) and 20 μM Aβ 42 were added and co-incubated for 24 h, and the cell survival rate was determined by the CCK-8 method.

[0081] The protective effects of CA-CDs and EDA-CDs on Aβ 42 induced cytotoxicity are shown as follows. Since the cell survival rates of CA-CDs and EDA-CDs were still above 85% and 90% respectively when the concentration was as high as 600 μg / mL, it indicated that the cytotoxicity of the two kinds of carbon dots was relatively small. And when the concentration of CA-CDs was less than 200 μg / mL, it had the effect of reducing Aβ Figure 9 cytotoxicity, which could increase the cell survival rate from <65% to above 93%. EDA-CDs could increase the cell survival rate to above 80% in the range of 50 - 200 μg / mL, indicating that both CA-CDs and EDA-CDs could inhibit Aβ 42 induced cytotoxicity and had potential therapeutic application prospects. 42 It is easy for those skilled in the art to understand that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0082] It is easy for those skilled in the art to understand that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of carbon dots in the red fluorescence turn-on imaging detection for protein aggregates, the carbon dots are used for preparing a protein aggregate detection reagent, the excitation wavelength of the detection is 510 nm, and the emission wavelength range is 520 - 800 nm; the carbon dots are prepared through the following steps: (1) Dissolve ethylenediamine, congo red and an oxidant in deionized water, heat for reaction; then centrifuge to collect the supernatant; the oxidant contains a peroxide bond; (2) Dialyze the supernatant obtained in step (1), take out the internal dialysis solution after dialysis, and heat to obtain a carbon dot concentrate.

2. Application of carbon dots in the preparation of a protein aggregate inhibitor drug, the carbon dots are prepared through the following steps: (1) Dissolve ethylenediamine, congo red and an oxidant in deionized water, heat for reaction; then centrifuge to collect the supernatant; the oxidant contains a peroxide bond; (2) Dialyze the supernatant obtained in step (1), take out the internal dialysis solution after dialysis, and heat to obtain a carbon dot concentrate.

3. The application according to claim 1 or 2, characterized in that, The protein aggregate is an aggregate containing a β-sheet structure formed by aggregating proteins.

4. The application according to claim 3, characterized in that The aggregating protein is at least one of β-amyloid protein, α-synuclein, human islet amyloid polypeptide, insulin, huntingtin, superoxide dismutase, prion protein and lysozyme.

5. The application according to claim 1 or 2, characterized in that, The oxidant is at least one of ammonium persulfate, benzoyl peroxide and hydrogen peroxide.

6. The application according to claim 1 or 2, characterized in that, The concentration of ethylenediamine in the deionized water is 0.2 - 3 mol / L, the oxidant concentration is 0.002 - 0.5 mol / L, and the congo red concentration is 0.2 - 20 g / L.

7. The application according to claim 1 or 2, characterized in that, The heating temperature is 100 - 300 °C, and the time is 5 min - 8 h.

8. Application of carbon dots in the preparation of a reactive oxygen species scavenging drug, the carbon dots are prepared through the following steps: (1) Dissolve ethylenediamine, congo red and an oxidant in deionized water, heat for reaction; then centrifuge to collect the supernatant; the oxidant contains a peroxide bond; (2) Dialyze the supernatant obtained in step (1), take out the internal dialysis solution after dialysis, and heat to obtain a carbon dot concentrate.

9. The application according to claim 8, characterized in that The oxidant is at least one of ammonium persulfate, benzoyl peroxide and hydrogen peroxide.

10. The application according to claim 8, characterized in that, The concentration of ethylenediamine in the deionized water is 0.2 - 3 mol / L, the oxidant concentration is 0.002 - 0.5 mol / L, and the congo red concentration is 0.2 - 20 g / L.

11. The application according to claim 8, wherein The heating temperature is 100 - 300 °C, and the time is 5 min - 8 h.

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