A quantitative detection method for fluorescent carbon quantum dots

Abstract

The present invention provides a quantitative detection method for fluorescent nano-carbon quantum dots. It comprises the following steps: preparing a sample solution to be tested and detecting the fluorescence intensity; preparing a reference solution with gradient concentrations using cresyl violet or rhodamine as a reference and detecting the corresponding fluorescence intensity, then establishing a standard curve to obtain a fitting equation; substituting the fluorescence intensity of the sample solution to be tested into the fitting equation to obtain a concentration value, and then calculating the percentage of the concentration value to the concentration of the sample solution to be tested, which is the carbon dot content. The detection method provided by the present invention can quantitatively detect the carbon dot content in vitro, in cells, and in tissues, with high accuracy, good repeatability, high precision, and high recovery rate, breaking through the bottleneck that there is no reference substance for carbon dots and it is difficult to perform quantitative detection, and providing a quantitative research method for the establishment of carbon dot quality standards and in-depth research in biological diagnosis and treatment.

Description

Technical Field

[0001] The invention belongs to the field of detection methods, and specifically relates to a quantitative detection method for fluorescent carbon quantum dots. Background Art

[0002] Carbon quantum dots (CDs) are inert nanoparticles with a size of less than 10nm and self-luminous. Due to their excellent fluorescence properties, they are also called nano fluorescent carbon dots. Carbon dots have a history of nearly 20 years, and their concept has been gradually expanded. At present, carbon dots have become a general term for a class of fluorescent carbon nanomaterials. Compared with organic fluorescent dyes and traditional semiconductor quantum dots, the emission wavelength of carbon dots is adjustable, and their carbon components make them less toxic to organisms and have fewer adverse reactions. These excellent properties make them have good application potential in imaging. At present, carbon dots have been widely studied and applied in ion molecule detection, bioimaging, photocatalysis, optical materials, etc., especially in the biomedical field. Showing strong development potential. At the same time, due to the presence of functional groups that are easy to modify on the surface of carbon dots, this feature provides the possibility of cancer cell labeling and tracing, and provides a new research direction for the realization of visual detection. While the research on carbon dot cell imaging is gradually maturing, the biocompatibility and biomembrane penetration of carbon dots are gradually developing and becoming a new research focus for the treatment of drug-resistant bacteria and drug-resistant tumors. In view of the huge potential prospects of carbon dots in the fields of medical diagnosis and treatment and new drug research and development, the dose-effect relationship of carbon dots requires more complete research on the quality standards of carbon dots. The establishment of a method for determining the content of carbon dots, especially a quantitative method for carbon dots in vivo, is bound to become the basis for further research on carbon dots.

[0003] Since carbon dots are composed of a group of zero-dimensional aggregate particles with a particle size below 10nm, most quantitative studies on fluorescent nanomaterials use "rough quantification", such as rough judgment based on subjective visual brightness, which is often inaccurate. However, for the future goal of accurate diagnosis and treatment, fluorescent nanomaterials that serve humans and other biological objects require more accurate quality control to ensure the reproducibility of efficacy and safety. The establishment of a method for determining the content of nanomaterials is one of the important research projects for the establishment of carbon dot quality standards, and is a basic condition for achieving drug safety, effectiveness, and quality control.

[0004] Since there is no pure substance as a reference substance for carbon dots, the establishment of a quantitative method for carbon dots has always been a topic to be developed, and there is no report on a method for quantitative detection of carbon dots. If we can break through the bottleneck of no reference substance for carbon dots and establish a quantitative carbon dot content determination method, it will be of great significance. Summary of the invention

[0005] The purpose of the present invention is to use pure fluorescent substances as reference substances, and to provide a method for content determination for the establishment of carbon dot quality standards and in vivo quantitative detection by establishing a fluorescence spectrophotometric method for content determination of nano fluorescent carbon dots in vitro and in vivo.

[0006] The present invention provides a quantitative detection method for fluorescent carbon quantum dots, comprising the following steps:

[0007] (1) preparing a sample solution with a concentration of C0 using a diluent, detecting the fluorescence intensity, and obtaining the fluorescence intensity F of the sample;

[0008] (2) Prepare a stock solution of the reference substance with the diluent. Dilute the stock solution with the diluent to obtain at least 5 reference substance solutions with gradient concentrations of C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C22, C12, C13, C14, C15, C16, C17, C18, C29, C21, C21, C30, C19 n , detect the fluorescence intensity, and obtain the fluorescence intensity F1, F2…F corresponding to the gradient concentration of the reference solution n , n is an integer greater than or equal to 5;

[0009] (3) Using the concentrations of the reference solution C1, C2, ... C n and the corresponding fluorescence intensities F1, F2…F n Draw a standard curve and obtain the fitting equation of the fluorescence intensity-concentration of the reference substance by linear fitting;

[0010] (4) Substituting the fluorescence intensity F of the sample to be tested measured in step (1) into the fitting equation obtained in step (3) to calculate the concentration C';

[0011] (5) Calculate the carbon quantum dot content C in the sample to be tested according to the following formula: C% = (C' / C0)*100%;

[0012] Wherein, the reference substance is cresyl purple or rhodamine.

[0013] Furthermore, the fluorescent carbon quantum dots are DCCDs, FA-DCCDs, ACUCDs, FA-ACUCDs, UCPG-Fe or FA-UCPG-Fe;

[0014] The DCCDs are carbon quantum dots prepared from citric acid, urea, phosphoric acid and N,N-dimethylformamide as raw materials, and the FA-DCCDs are tumor-targeted carbon quantum dots formed by chemically bonding DCCDs and folic acid; the ACUCDs are carbon quantum dots prepared from ammonium citrate and urea as raw materials, and the FA-ACUCDs are tumor-targeted carbon quantum dots formed by chemically bonding ACUCDs and folic acid; the UCPG-Fe are carbon quantum dots prepared from citric acid, urea, PEG400 and iron salts as raw materials, and the FA-UCPG-Fe are tumor-targeted carbon quantum dots formed by chemically bonding UCPG-Fe and folic acid.

[0015] Furthermore, the sample to be tested is a solid mixture or tissue fluid containing fluorescent carbon quantum dots, or a lysate obtained by lysing cells that have taken up fluorescent carbon quantum dots; preferably, the tissue fluid is liver tissue fluid or kidney tissue fluid.

[0016] Furthermore, the above diluent is ultrapure water or sodium chloride solution. Preferably, the concentration of the sodium chloride solution is 0.9%wt.

[0017] Furthermore, the above-mentioned detection of fluorescence intensity is: using an ELISA instrument to detect the fluorescence intensity at an emission wavelength λm under an excitation wavelength λ; the emission wavelength λm is the maximum emission wavelength of the fluorescent carbon quantum dots, and the excitation wavelength λ is the excitation wavelength corresponding to the fluorescent carbon quantum dots when the maximum emission wavelength is obtained.

[0018] Furthermore, the fluorescent carbon quantum dots are DCCDs, the reference substance is cresyl violet; the fluorescence intensity is detected at an excitation wavelength of 570 nm and an emission wavelength of 634 nm. Preferably, the sample to be tested is a solid mixture containing fluorescent carbon quantum dots, the diluent is ultrapure water, the range of C0 is 1.250 to 19.99 μg / mL, C1, C2…C n The range is 0.3900-12.50 μg / mL;

[0019] Or, the sample to be tested is a lysate obtained by lysing cells that have taken up fluorescent carbon quantum dots, the diluent is a sodium chloride solution, the range of C0 is 0.1000-1.602 μg / mL, C1, C2...C n The range is 0.0250~0.8010μg / mL;

[0020] Or, the sample to be tested is liver tissue fluid containing fluorescent carbon quantum dots, the diluent is sodium chloride solution, the range of C0 is 1.250-19.94 μg / mL, C1, C2...C n The range is 0.3906-6.250 μg / mL;

[0021] Or, the sample to be tested is renal tissue fluid containing fluorescent carbon quantum dots, the diluent is sodium chloride solution, the range of C0 is 0.6231-9.970 μg / mL, C1, C2...C n The range is 0.1900~3.120μg / mL.

[0022] Furthermore, the fluorescent carbon quantum dots are FA-DCCDs, the reference substance is rhodamine; the fluorescence intensity is the fluorescence intensity at an emission wavelength of 560nm detected under an excitation wavelength of 460nm; preferably, the sample to be tested is a solid mixture containing fluorescent carbon quantum dots, the diluent is ultrapure water, the range of C0 is 2.005-32.08μg / mL, C1, C2…C n The range is 0.8500~13.60μg / mL;

[0023] Or, the sample to be tested is a lysate obtained by lysing cells that have taken up fluorescent carbon quantum dots, the diluent is a sodium chloride solution, the range of C0 is 0.4988-7.980 μg / mL, C1, C2...C n The range is 0.2262~3.620μg / mL.

[0024] Furthermore, the fluorescent carbon quantum dots are ACUCDs, the reference substance is rhodamine; the fluorescence intensity is detected at an excitation wavelength of 490 nm and an emission wavelength of 570 nm; preferably, the sample to be tested is a solid mixture containing fluorescent carbon quantum dots, the diluent is ultrapure water, the range of C0 is 0.9850 to 15.74 μg / mL, C1, C2…C n The range is 0.4300~6.802μg / mL.

[0025] Furthermore, the carbon quantum dots are FA-ACUCDs, the reference substance is rhodamine; the fluorescence intensity is the fluorescence intensity at an emission wavelength of 534 nm detected under an excitation wavelength of 420 nm; preferably, the sample to be tested is a solid mixture containing fluorescent carbon quantum dots, the diluent is ultrapure water, the range of C0 is 7.520-120.3 μg / mL, C1, C2…C n The range is 1.700~27.20μg / mL.

[0026] Furthermore, the carbon quantum dots are UCPG-Fe, the reference substance is rhodamine; the fluorescence intensity is detected at an excitation wavelength of 480 nm and an emission wavelength of 555 nm; preferably, the sample to be tested is a solid mixture containing fluorescent carbon quantum dots, the diluent is ultrapure water, the range of C0 is 0.7800 to 12.48 μg / mL, C1, C2…C n The range is 0.2124~3.398μg / mL.

[0027] Furthermore, the carbon quantum dots are FA-UCPG-Fe, the reference substance is rhodamine; the fluorescence intensity is the fluorescence intensity at an emission wavelength of 545 nm detected at an excitation wavelength of 480 nm; preferably, the sample to be tested is a solid mixture containing fluorescent carbon quantum dots, the diluent is ultrapure water, the range of C0 is 0.3000 to 4.801 μg / mL, C1, C2…C n The range is 0.2100~3.402μg / mL.

[0028] When developing and studying candidate carbon dots that can be used to establish fluorescent quantitative detection of DX1002 for the first time, both the preparation process and the establishment of quantitative methods will face great difficulties. In order to meet the application of biological imaging and photothermal therapy research, how to develop and prepare near-infrared luminescent carbon dots with high quantum yield is one of the difficulties in the early stage of work; because near-infrared light waves can better pass through biological tissues and can better distinguish from the interference of the organism's own blue fluorescence, near-infrared luminescent carbon dots are highly favored. The inventor screened a variety of carbon dots with different fluorescent properties prepared by different processes, and finally obtained 3 long-wavelength carbon dots (λem≥500nm) required for in vivo imaging, anti-resistant bacteria or anti-resistant tumors as candidate carbon dots with superior performance.

[0029] In addition, how to select the reference substance for carbon dot quantification is the second experimental difficulty. Since the preparation materials of carbon dots usually include carbon source, nitrogen source, solvent and other substances, and although the prepared carbon dots are inert nanoparticles, they are aggregates of particles of various particle sizes, it is difficult to prepare carbon dots of a single particle size as a reference substance, which is also one of the bottlenecks restricting the establishment of carbon dot quantitative methods. After a lot of exploration, the inventors found a suitable pure fluorescent reference substance as a substitute reference substance for carbon dots based on the fluorescence characteristics of the prepared carbon dots.

[0030] The applicant selected the appropriate pure fluorescent reference substance with the candidate carbon dots as the object, first established the in vitro quantitative determination method of the candidate carbon dots, and further realized the establishment of the quantitative method of the candidate carbon dots in cells and the establishment of the quantitative method of the candidate carbon dots in vivo tissues on the basis of determining that the carbon dots have a quantitative relationship. The present invention breaks through the bottleneck that there is no reference substance for carbon dots and it is difficult to conduct quantitative detection, and provides a quantitative research method for the establishment of carbon dot quality standards and in-depth research in biological diagnosis and treatment.

[0031] The "gradient concentration" mentioned in the present invention refers to: concentrations C1, C2, ... C n The relationship between is a fixed multiple relationship, that is, C2 / C1=C3 / C2=C4 / C3=…=C n / C n -1, n≥5.

[0032] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0033] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 (A) Linear relationship between DCCDs fluorescence intensity and concentration; (B) Linear relationship between cresyl violet fluorescence intensity and concentration.

[0035] Figure 2 (A) The linear relationship between the fluorescence intensity and concentration of FA-DCCDs; (B) The linear relationship between the fluorescence intensity and concentration of Rhodamine 6G.

[0036] Figure 3 (A) Linear relationship between the fluorescence intensity of ACUCDs and its concentration; (B) Linear relationship between the fluorescence intensity of Rhodamine 6G and its concentration.

[0037] Figure 4 (A) The linear relationship between the fluorescence intensity and concentration of FA-ACUCDs; (B) The linear relationship between the fluorescence intensity and concentration of Rhodamine 6G.

[0038] Figure 5 (A) The linear relationship between the fluorescence intensity of UCPG-Fe and its concentration; (B) The linear relationship between the fluorescence intensity of Rhodamine 6G and its concentration.

[0039] Figure 6 (A) The linear relationship between the fluorescence intensity of FA-UCPG-Fe and its concentration; (B) The linear relationship between the fluorescence intensity of Rhodamine 6G and its concentration.

[0040] Figure 7 (A) The effect of cell environment on DCCDs; (B) The effect of cell environment on FA-DCCDs.

[0041] Figure 8 (A) The linear relationship between the fluorescence intensity of DCCDs and its concentration in Hela cells; (B) The linear relationship between the fluorescence intensity of cresyl violet and its concentration in Hela cells.

[0042] Fig. 9 (A) The linear relationship between the fluorescence intensity and concentration of FA-DCCDs in Hela cells; (B) The linear relationship between the fluorescence intensity and concentration of Rhodamine 6G in Hela cells.

[0043] Fig.10 (A) Fluorescence emission spectra of DCCDs, liver tissue fluid, and DCCDs-liver tissue fluid; (B) Fluorescence emission spectra of DCCDs, kidney tissue fluid, and DCCDs-kidney tissue fluid; (C) Fluorescence emission spectra of DCCDs, DCCDs-liver tissue fluid, and DCCDs-kidney tissue fluid.

[0044] Fig.11 (A) The linear relationship between the fluorescence intensity and concentration of DCCDs in liver tissue fluid; (B) The linear relationship between the fluorescence intensity and concentration of cresyl violet in liver tissue fluid.

[0045] Fig.12 (A) The linear relationship between the fluorescence intensity of DCCDs and its concentration in renal tissue fluid; (B) The linear relationship between the fluorescence intensity of cresyl violet and its concentration in renal tissue fluid. DETAILED DESCRIPTION

[0046] Unless otherwise specified, the raw materials and equipment used in the present invention are all known products obtained by purchasing commercially available products.

[0047] The preparation method of the carbon dots used in the embodiments of the present invention is as follows:

[0048] 1. Preparation of DCCDs:

[0049] Weigh 2.0g of citric acid and 1.0g of urea respectively and place them in a beaker, then add 2ml of dopant phosphoric acid and 10ml of DMF, stir and dissolve with ultrasound. Transfer the solution to a 50ml reactor liner (polytetrafluoroethylene), reinforce it with a metal shell of the reactor, and react at 180°C for 10h. Filter the carbon dot stock solution, centrifuge (13000r / min, 10min), dialyze (500Da, 24h), and evaporate the solution with an oil pump to obtain carbon dot solid.

[0050] After testing, the DCCDs solution showed deep red under sunlight and emitted blue fluorescence under 365nm ultraviolet light excitation. The emission wavelength of DCCDs was relatively stable, with only a slight red shift as the excitation wavelength increased. It had a maximum emission wavelength of 634nm at an excitation wavelength of 570nm, and the maximum emission wavelength was located in the red light region. After preliminary cell imaging tests, the carbon dots had good imaging capabilities.

[0051] 2. Preparation of FA-DCCDs:

[0052] (1) Preparation of active folate: 88.28 mg of folic acid, 115.02 mg of EDC, and 46.04 mg of NHS were accurately weighed into a round-bottom flask, and 4 mL of PBS (pH 7.4) was added. The mixture was shaken thoroughly until completely dissolved, and stirred continuously on a magnetic stirrer overnight.

[0053] (2) Preparation of FA-DCCDs tumor-targeted carbon quantum dots: Accurately weigh 88.28 mg of DCCDs in a microcentrifuge tube, add 2 mL of PBS (7.4) to dissolve, and slowly add dropwise to 2 mL of stirred folic acid active ester. After continuous stirring for 24 h, the reaction solution is dialyzed with PBS buffer and ultrapure water (MWCO = 500 Da, 24 h), and freeze-dried to obtain.

[0054] After testing, the FA-DCCDs solution showed orange-red color under sunlight and visual blue fluorescence under ultraviolet light. FA-DCCDs has non-excitation-dependent characteristics and has a maximum emission wavelength of 560nm at an excitation wavelength of 460nm. The maximum emission wavelength is located in the yellow-green light region, and after preliminary cell imaging tests, it can be used for subsequent cell imaging research.

[0055] 3. Preparation of ACUCDs:

[0056] Weigh 1 g of ammonium citrate and 1 g of urea in a mortar and grind them thoroughly until mixed. Then transfer the mixture into a crucible and heat it in a resistance furnace at 150°C for 10 hours. After the reaction is completed, let it naturally cool to room temperature, add 40 mL of ultrapure water and perform ultrasonic-assisted extraction. Filter the extract, and then dialyze the filtrate (500Da, 24 hours) and freeze-dry to obtain carbon dot solid.

[0057] The ACUCDs solution was tested to be pale yellow in sunlight and emitted yellow-green fluorescence under 365nm ultraviolet light excitation. ACUCDs has excitation-independent characteristics, with a maximum emission wavelength of 570nm at an excitation wavelength of 490nm, and the maximum emission wavelength is located in the yellow-green light region. After preliminary cell imaging tests, the carbon dots have potential for imaging applications.

[0058] 4. Preparation of FA-ACUCDs:

[0059] (1) Preparation of active folate: 88.28 mg of folic acid, 115.02 mg of EDC, and 46.04 mg of NHS were accurately weighed in a round-bottom flask, and 4 mL of PBS (pH = 7.4) was added. The mixture was shaken thoroughly to dissolve completely, and stirred continuously on a magnetic stirrer overnight to activate FA.

[0060] (2) Preparation of FA-ACUCDs tumor-targeting carbon quantum dots: 88.28 mg ACUCDs were accurately weighed in a microcentrifuge tube, dissolved in 2 mL PBS (pH = 7.4), and slowly added dropwise to 2 mL of stirred folic acid active ester. The pH was then adjusted to 9 with ethylenediamine. After continuous stirring for 24 h, the reaction solution was dialyzed with PBS buffer and ultrapure water (MWCO = 500 Da, 24 h), and freeze-dried to obtain the product.

[0061] After testing, the FA-ACUCDs solution appears yellow under sunlight and shows visual green fluorescence under ultraviolet light. FA-ACUCDs is stable at an emission wavelength of 534nm, and the excitation wavelength is 420nm. The maximum emission wavelength is in the green light region. After initial cell imaging tests, it can be used for subsequent cell imaging studies.

[0062] 5. Preparation of UCPG-Fe:

[0063] Weigh 1.0g citric acid and 2.0g urea respectively and place them in a mortar, add 5ml water and 15ml PEG400 successively for grinding, and add 0.5g ferric chloride hexahydrate and continue grinding until uniform after being evenly dispersed. Transfer the sample to the inner liner of the reactor (polytetrafluoroethylene), reinforce it with the metal shell of the reactor, react at 240℃ for 3h, extract it with acetone twice, filter it, and dry it to obtain the carbon dot solid.

[0064] After testing, the UCPG-Fe solution appears red under sunlight and emits white fluorescence under 365nm excitation of ultraviolet light. Its visual red may be the color of the iron ions themselves. UCPG-Fe has the characteristics of excitation light dependence. It has a maximum emission wavelength of 555nm at an excitation wavelength of 480nm, and the maximum emission wavelength is located in the green light region. The carbon dots have strong absorption in the range of 550-600nm and can be considered for cell imaging. After initial cell imaging tests, the carbon dots have good imaging capabilities.

[0065] 6. Preparation of FA-UCPG-Fe:

[0066] (1) Preparation of active folate: 88.28 mg of folic acid, 115.02 mg of EDC, and 46.04 mg of NHS were accurately weighed into a round-bottom flask, and 4 mL of PBS (pH 7.4) was added. The mixture was shaken thoroughly to dissolve completely, and stirred continuously on a magnetic stirrer overnight to activate FA.

[0067] (2) Preparation of FA-UCPG-Fe tumor-targeted carbon quantum dots: Accurately weigh 88.28 mg of UCPG-Fe in a microcentrifuge tube, add 2 mL of PBS (7.4) to dissolve, slowly add dropwise to 2 mL of stirred folic acid active ester, then add dropwise ethylenediamine to adjust the pH to 9. After continuous stirring for 24 h, the reaction solution was dialyzed with PBS buffer and ultrapure water (MWCO = 500 Da, 24 h), and freeze-dried to obtain the product.

[0068] After testing, the FA-UCPG-Fe solution showed a light yellow color under sunlight and a visual white fluorescence under ultraviolet light. FA-UCPG-Fe has an excitation-dependent characteristic, with a maximum emission wavelength of 545nm at an excitation wavelength of 480nm, and the maximum emission wavelength is located in the green light region. After the initial test of cell imaging, it can be used for subsequent cell imaging research.

[0069] Example 1. Establishment of an in vitro quantitative detection method for carbon dots and their folic acid carbon dot pairs

[0070] 1. Preparation of stock solution

[0071] 1.1 Preparation of reference stock solution

[0072] Accurately weigh 0.1002 g of cresol purple, add ultrapure water to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 1.002 mg / mL cresol purple reference solution. Set aside.

[0073] Accurately weigh 0.1088 g of Rhodamine 6G, add ultrapure water to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 1.088 mg / mL Rhodamine 6G reference solution. Set aside.

[0074] 1.2 Preparation of stock solutions of carbon dots and their folic acid conjugates

[0075] Accurately weigh 0.1280 g DCCDs, add ultrapure water to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 1.280 mg / mL DCCDs solution. Set aside.

[0076] Accurately weigh 0.1007 g ACUCDs, add ultrapure water to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 1.007 mg / mL ACUCDs solution. Set aside.

[0077] Accurately weigh 0.9986 g UCPG-Fe, add ultrapure water to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 0.9986 mg / mL UCPG-Fe solution. Set aside.

[0078] Accurately weigh 0.1002 g FA-DCCDs, add ultrapure water to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 0.1002 g / mL FA-DCCDs-FA solution. Set aside.

[0079] Accurately weigh 0.1925 g FA-ACUCDs, add ultrapure water to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 1.925 mg / mL FA-DCCDs solution. Set aside.

[0080] Accurately weigh 0.9604 g FA-UCPG-Fe, add ultrapure water to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 0.9604 mg / mL FA-UCPG-Fe solution. Set aside.

[0081] 2. Establishment of DCCDs quantitative method

[0082] 2.1 Determination method

[0083] 100 μL of each test solution was added to a 96-well cell culture plate (hereinafter referred to as 96-well plate), and fluorescence was measured using an ELISA reader at a wavelength of (excitation wavelength / maximum emission wavelength) Ex 570 nm / Em 634 nm.

[0084] 2.2 Methodological validation

[0085] 2.2.1 Linearity and range

[0086] 2.2.1.1 Investigation of linear relationship of DCCDs

[0087] Take appropriate amount of DCCDs stock solution to prepare 1.250, 2.499, 4.998, 9.996, 19.99 μg / mL DCCDs solutions respectively. Perform the determination according to the method under “2.1 Determination Method”.

[0088] Depend on Figure 1 A shows that DCCDs has a good linear relationship in the range of 1.250 to 19.99 μg / mL (y = 5.293x-1.9743, y is the fluorescence intensity (au), x is the concentration (μg / mL), the same below. R 2 =0.9997).

[0089] 2.2.1.2 Investigation of the linear relationship of cresyl violet

[0090] Take an appropriate amount of cresyl violet reference stock solution to prepare 0.3900, 0.7800, 1.560, 3.125, 6.250, and 12.50 μg / mL cresyl violet reference solutions, respectively. Perform the determination according to the method under “2.1 Determination Method”.

[0091] Depend on Figure 1 B shows that cresol violet has a good linear relationship in the range of 0.3900 to 12.50 μg / mL: (y = 17.235x-7.7131, R 2 =0.9978).

[0092] It can be seen that DCCDs and cresyl violet each have a good linear relationship within a certain concentration range, indicating that there is a quantitative relationship between the concentration of DCCDs and cresyl violet and their luminescence intensity at a specified wavelength, and on this basis, the fluorescence intensity range of DCCDs is within the linear range of cresyl violet, which can ensure that the quantification within the concentration range of cresyl violet can cover the quantitative range of DCCDs.

[0093] 2.3 Quantitative detection:

[0094] Dilute the DCCDs stock solution to a concentration of C0 with ultrapure water, C0 is in the range of 1.250-19.99 μg / mL, and detect the fluorescence intensity according to "2.1 Determination Method" to obtain the fluorescence intensity F of the sample to be tested;

[0095] Substitute the measured fluorescence intensity F of the sample to be tested into the linear fitting equation of the cresyl violet reference substance: y = 17.235x-7.7131, take y = F, and calculate the value of the concentration C' = x;

[0096] (5) The carbon quantum dot content C in the sample to be tested is calculated according to the following formula: C%=(C' / C0)*100%.

[0097] 3. Establishment of FA-DCCDs quantitative method

[0098] 3.1 Determination method

[0099] 100 μL of each test solution was added to a 96-well cell culture plate (hereinafter referred to as 96-well plate) and measured using an ELISA reader at a wavelength of Ex 460 nm / Em 560 nm.

[0100] 3.2 Methodological validation

[0101] 3.2.1 Linearity and range

[0102] 3.2.1.1 Investigation of the linear relationship between FA and DCCDs

[0103] Take the FA-DCCDs stock solution and prepare 2.005, 4.010, 8.02, 16.04, and 32.08 μg / mL FA-DCCDs solutions respectively. Perform the determination according to the method under "3.1 Determination Method".

[0104] Depend on Figure 2 A shows that FA-DCCDs has a good linear relationship in the range of 2.005-32.08 μg / mL (y=6.6001x+21.69, R 2 =0.9972).

[0105] 3.2.1.2 Investigation of the linear relationship of Rhodamine 6G

[0106] Take the Rhodamine 6G reference substance stock solution and prepare 0.8500, 1.700, 3.400, 6.800, and 13.60 μg / mL Rhodamine 6G reference substance solutions respectively. Perform the determination according to the method under "3.1 Determination Method".

[0107] Depend on Figure 2B shows that rhodamine 6G has a good linear relationship in the range of 0.8500 to 13.60 μg / mL (y = 46.02x-44.367, R 2 =0.9964).

[0108] It can be seen that FA-DCCDs and rhodamine 6G each have a good linear relationship within a certain concentration range, indicating that there is a quantitative relationship between the concentration of FA-DCCDs and rhodamine 6G and their luminescence intensity at a specified wavelength, and on this basis, the fluorescence intensity range of FA-DCCDs is within the linear range of rhodamine 6G, which can ensure that the quantification within the concentration range of rhodamine 6G can cover the quantitative range of FA-DCCDs.

[0109] 3.3 Quantitative detection:

[0110] Dilute the stock solution of the sample FA-DCCDs to be tested with ultrapure water to a concentration C0, C0 is in the range of 2.005-32.08 μg / mL, and detect the fluorescence intensity according to "3.1 Determination Method" to obtain the fluorescence intensity F of the sample to be tested;

[0111] Substitute the measured fluorescence intensity F of the sample to be tested into the linear fitting equation of the rhodamine reference substance: y = 46.02x-44.367, take y = F, and calculate the value of the concentration C' = x;

[0112] (5) The carbon quantum dot content C in the sample to be tested is calculated according to the following formula: C%=(C' / C0)*100%.

[0113] 4. Establishment of ACUCDs quantitative method

[0114] 4.1 Determination method

[0115] 100 μL of each test solution was added to a 96-well cell culture plate (hereinafter referred to as 96-well plate) and measured using an ELISA reader with an excitation wavelength of 490 nm and an emission wavelength of 570 nm.

[0116] 4.2 Methodological validation

[0117] 4.2.1 Linearity and range

[0118] 4.2.1.1 Investigation of the linear relationship of ACUCDs

[0119] Accurately measure an appropriate amount of ACUCDs stock solution to prepare 0.9850, 1.965, 3.935, 7.865, and 15.74 μg / mL ACUCDs solutions, respectively. Perform the assay according to the method under “4.1 Assay Method”.

[0120] Depend on Figure 3A shows that ACUCDs has a good linear relationship in the range of 0.9850-15.74 μg / mL (y=8.6123x+7.4863, R 2 =0.994).

[0121] 4.2.1.2 Investigation of the linear relationship of Rhodamine 6G

[0122] Accurately measure an appropriate amount of Rhodamine 6G reference stock solution to prepare 0.4300, 0.8500, 1.700, 3.401, and 6.802 μg / mL cresyl purple reference solutions, respectively. Perform the determination according to the method under “4.1 Determination Method”.

[0123] Depend on Figure 3 B shows that rhodamine 6G has a good linear relationship in the range of 0.4300 to 6.802 μg / mL (y = 68.505x-37.183, R 2 =0.9966).

[0124] In summary, ACUCDs and rhodamine 6G each have a good linear relationship within a certain concentration range, indicating that there is a quantitative relationship between the concentration of ACUCDs and rhodamine 6G and their luminescence intensity at a specified wavelength, and on this basis, the fluorescence intensity range of ACUCDs is within the linear range of rhodamine 6G, which can ensure that the quantification within the concentration range of rhodamine 6G can cover the quantitative range of ACUCDs.

[0125] 4.3 Quantitative detection:

[0126] Dilute the stock solution of the ACUCDs sample to be tested with ultrapure water to a concentration C0, C0 is in the range of 0.9850-15.74 μg / mL, and detect the fluorescence intensity according to "4.1 Determination Method" to obtain the fluorescence intensity F of the sample to be tested;

[0127] Substitute the measured fluorescence intensity F of the sample to be tested into the linear fitting equation of the rhodamine reference substance: y = 68.505x-37.183, take y = F, and calculate the value of the concentration C' = x;

[0128] (5) The carbon quantum dot content C in the sample to be tested is calculated according to the following formula: C%=(C' / C0)*100%.

[0129] 5. Establishment of FA-ACUCDs quantitative method

[0130] 5.1 Determination method

[0131] 100 μL of each test solution was added to a 96-well cell culture plate (hereinafter referred to as 96-well plate), and fluorescence was measured using an enzyme reader with an excitation wavelength of 420 nm and an emission wavelength of 534 nm.

[0132] 5.2 Methodological validation

[0133] 5.2.1 Linearity and range

[0134] 5.2.1.1 Investigation of the linear relationship between FA and ACUCDs

[0135] Accurately measure an appropriate amount of FA-ACUCDs stock solution to prepare 7.520, 15.04, 30.08, 60.16, and 120.3 μg / mL FA-ACUCDs solutions, respectively. Perform the determination according to the method under "5.1 Determination Method".

[0136] From 4A, we can see that FA-ACUCDs has a good linear relationship in the range of 7.520 to 120.3 μg / mL (y = 1.2251x + 7.9835, R 2 =0.9928).

[0137] 5.2.1.2 Investigation of the linear relationship of Rhodamine 6G

[0138] Accurately measure the stock solution of Rhodamine 6G reference substance, and prepare 1.700, 3.400, 6.800, 13.60, and 27.20 μg / mL of Rhodamine 6G reference substance solutions respectively. Perform the determination according to the method under "5.1 Determination Method".

[0139] Depend on Figure 4 B shows that rhodamine 6G has a good linear relationship in the range of 1.700 to 27.20 μg / mL (y = 8.2362x-9.5705, R 2 =0.9973).

[0140] In summary, FA-ACUCDs and rhodamine 6G each have a good linear relationship within a certain concentration range, indicating that there is a quantitative relationship between the concentration of FA-ACUCDs and rhodamine 6G and their luminescence intensity at a specified wavelength, and on this basis, the fluorescence intensity range of FA-ACUCDs is within the linear range of rhodamine 6G, which can ensure that the quantification within the concentration range of rhodamine 6G can cover the quantitative range of FA-ACUCDs.

[0141] 5.3 Quantitative detection:

[0142] Dilute the stock solution of the sample FA-ACUCDs to be tested with ultrapure water to a concentration C0, C0 is in the range of 7.520-120.3 μg / mL, and detect the fluorescence intensity according to "5.1 Determination Method" to obtain the fluorescence intensity F of the sample to be tested;

[0143] Substitute the measured fluorescence intensity F of the sample to be tested into the linear fitting equation of the rhodamine reference substance: y = 8.2362x-9.5705, take y = F, and calculate the value of the concentration C' = x;

[0144] (5) The carbon quantum dot content C in the sample to be tested is calculated according to the following formula: C%=(C' / C0)*100%.

[0145] 6. Establishment of UCPG-Fe quantitative method

[0146] 6.1 Determination method

[0147] 100 μL of each test solution was added to a 96-well cell culture plate (hereinafter referred to as 96-well plate) and measured using an enzyme reader with an excitation wavelength of 480 nm and an emission wavelength of 555 nm.

[0148] 6.2 Methodological Validation

[0149] 6.2.1 Linearity and range

[0150] 6.2.1.1 Investigation of the linear relationship between UCPG and Fe

[0151] Take an appropriate amount of UCPG-Fe stock solution to prepare 0.7800, 1.560, 3.120, 6.240, and 12.48 μg / mL UCPG-Fe solutions, respectively. Perform the determination according to the method under "6.1 Determination Method".

[0152] From 5A, we can see that UCPG-Fe has a good linear relationship in the range of 0.7800 to 12.48 μg / mL (y = 0.4149x-0.097, R 2 =0.9998).

[0153] 6.2.1.2 Investigation of the linear relationship of Rhodamine 6G

[0154] Take the Rhodamine 6G reference stock solution and prepare 0.2124, 0.4248, 0.8495, 1.699, and 3.398 μg / mL cresyl violet reference solutions respectively. Perform the determination according to the method under "6.1 Determination Method".

[0155] Depend on Figure 5 B shows that there is a good linear relationship in the range of 0.2124 to 3.398 μg / mL (y = 37.961x-10.664, R 2 =0.9967).

[0156] In summary, UCPG-Fe and Rhodamine 6G each have a good linear relationship within a certain concentration range, indicating that there is a quantitative relationship between the concentration of UCPG-Fe and Rhodamine 6G and their luminescence intensity at a specified wavelength, and on this basis, the fluorescence intensity range of UCPG-Fe is within the linear range of Rhodamine 6G, which can ensure that the quantification within the concentration range of Rhodamine 6G can cover the quantitative range of UCPG-Fe.

[0157] 6.3 Quantitative detection:

[0158] Dilute the stock solution of the sample UCPG-Fe to a concentration of C0 with ultrapure water, C0 is in the range of 0.7800 to 12.48 μg / mL, and detect the fluorescence intensity according to "6.1 Determination Method" to obtain the fluorescence intensity F of the sample to be tested;

[0159] Substitute the measured fluorescence intensity F of the sample to be tested into the linear fitting equation of the rhodamine reference substance: y = 37.961x-10.664, take y = F, and calculate the value of the concentration C' = x;

[0160] (5) The carbon quantum dot content C in the sample to be tested is calculated according to the following formula: C%=(C' / C0)*100%.

[0161] 7. Establishment of FA-UCPG-Fe quantitative method

[0162] 7.1 Determination method

[0163] 100 μL of each test solution was added to a 96-well cell culture plate (hereinafter referred to as 96-well plate) and measured using an ELISA reader with an excitation wavelength of 480 nm and an emission wavelength of 545 nm.

[0164] 7.2 Methodological Validation

[0165] 7.2.1 Linearity and range

[0166] 7.2.1.1 Investigation of the linear relationship between FA-UCPG-Fe

[0167] Accurately measure an appropriate amount of FA-UCPG-Fe stock solution to prepare 0.3000, 0.6000, 1.200, 2.400, and 4.801 μg / mL FA-UCPG-Fe solutions, respectively.

[0168] Depend on Figure 6 A shows that FA-UCPG-Fe has a good linear relationship in the range of 0.3000-4.801 μg / mL (y=4.3441x-0.335, R2=0.9989).

[0169] 7.2.1.2 Investigation of the linear relationship of Rhodamine 6G

[0170] Accurately measure an appropriate amount of Rhodamine 6G reference stock solution to prepare 0.2100, 0.4200, 0.850, 1.701, and 3.402 μg / mL cresyl purple reference solutions, respectively. Perform the determination according to the method under “7.1 Determination Method”.

[0171] Depend on Figure 6 B shows that rhodamine 6G has a good linear relationship in the range of 0.2100-3.402 μg / mL (y=43.049x-13.659, R2=0.9924).

[0172] In summary, FA-UCPG-Fe and Rhodamine 6G each have a good linear relationship within a certain concentration range, indicating that there is a quantitative relationship between the concentrations of FA-UCPG-Fe and Rhodamine 6G and their luminescence intensity at a specified wavelength, and on this basis, the fluorescence intensity range of FA-UCPG-Fe is within the linear range of Rhodamine 6G, which can ensure that the quantification within the concentration range of Rhodamine 6G can cover the quantitative range of FA-UCPG-Fe.

[0173] 7.3 Quantitative detection:

[0174] Dilute the stock solution of the sample UCPG-Fe to a concentration of C0 with ultrapure water, C0 is in the range of 0.3000 to 4.801 μg / mL, and detect the fluorescence intensity according to "7.1 Determination Method" to obtain the fluorescence intensity F of the sample to be tested;

[0175] Substitute the measured fluorescence intensity F of the sample to be tested into the linear fitting equation of the rhodamine reference substance: y = 43.049x-13.659, take y = F, and calculate the value of the concentration C' = x;

[0176] (5) The carbon quantum dot content C in the sample to be tested is calculated according to the following formula: C%=(C' / C0)*100%.

[0177] Example 2: Establishment of the Intracellular Quantitative Detection Method of DCCDs and FA-DCCDs of the Present Invention

[0178] 1. Preparation of stock solution

[0179] 1.1 Preparation of reference stock solution

[0180] Accurately weigh 0.1003 g of cresyl purple, add sodium chloride solution (0.9%) to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 1.003 mg / mL cresyl purple reference solution. Set aside.

[0181] Accurately weigh 0.1076 g of rhodamine 6G, add sodium chloride solution (0.9%) to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 1.076 mg / mL rhodamine 6G reference solution. Set aside.

[0182] 1.2 Preparation of DCCDs and FA-DCCDs stock solutions

[0183] Accurately weigh 0.1011 g DCCDs, add sodium chloride solution (0.9%) to dissolve, transfer the solution to a 100 mL volumetric flask, and adjust the volume to obtain a 1.011 mg / mL DCCDs solution. Set aside.

[0184] Accurately weigh 99.71 mg of FA-DCCDs, add sodium chloride solution (0.9%) to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 0.9971 mg / mL FA-DCCDs solution. Set aside.

[0185] 2. Effects of cell environment on fluorescent materials

[0186] Take 500 μL of DCCDs solution, add 500 ul of sodium chloride solution (0.9%), mix well, and obtain test solution-1; take 500 μL of cell solution lysed by DMSO, add 500 ul of sodium chloride solution (0.9%), mix well, and obtain test solution-2; take 500 μL of cell solution lysed by DMSO, add 500 μL of DCCDs solution, mix well, and obtain test solution-3. The three test solutions were scanned for fluorescence emission spectra at an excitation wavelength of 570 nm. The results are shown in Figure 7 A.

[0187] Take 500 μL of FA-DCCDs solution and sodium chloride solution (0.9%), mix well, and obtain test solution-4; take 500 μL of cell solution lysed by DMSO, add 500 ul of sodium chloride solution (0.9%), mix well, and obtain test solution-5; take 500 μL of cell solution lysed by DMSO, add 500 μL of FA-DCCDs solution, mix well, and obtain test solution-6. The three test solutions were scanned for fluorescence emission spectra at an excitation wavelength of 460 nm. The results are shown in Figure 7 B.

[0188] like Figure 7 As shown in A, under 570nm excitation, the cell fluid has no absorption peak in the emission light band of DCCDs, and the cell fluid has an enhancement effect on the fluorescence intensity of DCCDs. Therefore, a quantitative method for DCCDs can be established in a cell environment.

[0189] like Figure 7As shown in B, under 460nm excitation, the cell fluid has no absorption peak in the emission light band of FA-DCCDs, and the cell fluid has an enhancement effect on the fluorescence intensity of FA-DCCDs. Therefore, a quantitative method for FA-DCCDs can be established in a cell environment.

[0190] 3. Establishment of the intracellular quantitative method of DCCDs

[0191] 3.1 Determination principle and method

[0192] 3.1.1 Determination principle

[0193] Hela cells were cultured in a 96-well plate for 48 hours (cell content reached 90%), and then the culture was terminated. The culture medium (or extracellular carbon dot solution) was aspirated and washed three times with PBS (pH 7.30) to remove the residual culture medium (or extracellular carbon dot solution). 50 μL DMSO was added to each well for cell lysis to obtain cell fluid. 50 μL sodium chloride (0.9%) was added and fluorescence was measured using an enzyme-labeled instrument.

[0194] 3.1.2 Determination method

[0195] Hela cell line was used as the culture object, and the Hela cell line was cultured in a 96-well plate for 48 hours (cell content reached 90%), and the culture was terminated. The culture medium was aspirated, washed 3 times with PBS (pH7.30), 50 μL DMSO was added to each well, and 50 μL of each test solution was added to the 96-well cell plate, and the microplate reader was used for measurement at 570 nm excitation wavelength and 634 nm wavelength channel.

[0196] Methodological validation

[0197] 3.2.1 Linearity and range

[0198] 3.2.1.1 Investigation of linear relationship of DCCDs

[0199] Accurately measure an appropriate amount of DCCDs stock solution and prepare 0.1000, 0.2000, 0.4000, 0.8001, 1.201, and 1.602 μg / mL DCCDs solutions with sodium chloride solution (0.9%). Perform the determination according to the method under "3.1.2 Determination Method".

[0200] Depend on Figure 8 A shows that DCCDs has a good linear relationship in the range of 0.1000-1.602 μg / mL (y=8.2262x+0.5213, R2=0.9933).

[0201] 3.2.1.2 Investigation of the linear relationship of cresyl violet

[0202] Accurately measure an appropriate amount of cresyl violet reference stock solution, and prepare 0.0250, 0.0500, 0.1000, 0.2000, 0.4005, and 0.8010 μg / mL cresyl violet reference solutions with sodium chloride solution (0.9%). Perform the determination according to the method under "3.1.2 Determination method".

[0203] Depend on Figure 8 B shows that cresol violet has a good linear relationship in the range of 0.0250-0.8010 μg / mL (y=23.838x+0.4847, R 2 =0.9961).

[0204] In summary, the good linear relationship between intracellular DCCDs and cresyl violet within a certain concentration range indicates that there is a quantitative relationship between the concentration of DCCDs and cresyl violet and their luminescence intensity at a specified wavelength, and on this basis, the fluorescence intensity range of DCCDs is within the linear range of cresyl violet, which can ensure that the quantification within the concentration range of cresyl violet can cover the quantitative range of DCCDs.

[0205] 3.3. Quantitative detection:

[0206] Hela cell lines were used as culture objects. After the Hela cell lines were cultured in a 96-well plate for 36 hours, 10 mg / mL DCCD solution was intermittently administered at four experimental time points of 3 hours, 6 hours, 9 hours, and 12 hours, and the cells were taken up. The culture was terminated. The culture medium and carbon dot mixed culture medium were aspirated, and washed three times with PBS (pH 7.30) to wash away the residual culture medium and excess DCCD solution outside the cells. 50 μL DMSO was added to each well for cell lysis to obtain a cell solution containing DCCD, and 50 μL diluent: sodium chloride (0.9%) was added to obtain a test solution, wherein the concentration of DCCDs in the test sample (cell lysate) was C0, and the range of C0 was 0.1000-1.602 μg / mL. The fluorescence intensity was measured and detected using an ELISA instrument at an excitation wavelength of 570 nm and a wavelength channel of 634 nm to obtain the fluorescence intensity F of the test sample;

[0207] Substitute the measured fluorescence intensity F of the sample to be tested into the linear fitting equation of the cresyl violet reference substance: y=23.838x+0.4847, take y=F, and calculate the value of the concentration C'=x;

[0208] (5) The carbon quantum dot content C in the sample to be tested is calculated according to the following formula: C%=(C' / C0)*100%.

[0209] 4. Establishment of FA-DCCDs intracellular quantitative method

[0210] 4.1 Determination principle and method

[0211] 4.1.1 Determination principle

[0212] Hela cells were cultured in a 96-well plate for 48 hours (cell content reached 90%), and then the culture was terminated. The culture medium (or extracellular carbon dot solution) was aspirated and washed three times with PBS (pH 7.30) to remove the residual culture medium (or extracellular carbon dot solution). 50 μL DMSO was added to each well for cell lysis to obtain cell fluid. 50 μL sodium chloride (0.9%) was added and fluorescence was measured using an enzyme-labeled instrument.

[0213] 4.1.2 Determination method

[0214] Hela cells were cultured in a 96-well plate for 48 hours (cell content reached 90%), and then the culture was terminated. The culture medium was aspirated, washed 3 times with PBS (pH 7.30), 50 μL DMSO was added to each well, and 50 μL of each test solution was added to the 96-well cell plate, and the microplate reader was used for determination at 460 nm excitation wavelength and 560 nm wavelength channel.

[0215] Methodological validation

[0216] 4.2.1 Linearity and range

[0217] 4.2.1.1 Investigation of the linear relationship between FA and DCCDs

[0218] Take an appropriate amount of FA-DCCDs stock solution and prepare 0.4988, 0.9975, 1.995, 3.990, 5.990, and 7.980 μg / mL FA-DCCDs solutions with sodium chloride solution (0.9%). Perform the determination according to the method under "4.1.2 Determination Method".

[0219] Depend on Fig. 9 A shows that FA-DCCDs has a good linear relationship in the range of 0.4988 to 7.980 μg / mL (y = 2.0915x + 0.7486, R 2 =0.9968).

[0220] 4.2.1.2 Investigation of the linear relationship of Rhodamine 6G

[0221] Take an appropriate amount of Rhodamine 6G reference stock solution and prepare 0.2262, 0.4525, 0.9050, 1.810, 2.690, and 3.620 μg / mL Rhodamine 6G reference solutions with sodium chloride solution (0.9%). Perform the determination according to the method under "4.1.2 Determination method".

[0222] Depend on Fig. 9 B shows that rhodamine 6G has a good linear relationship in the range of 0.2262 to 3.620 μg / mL (y = 12.996x-2.9095, R 2 =0.9983).

[0223] In summary, the good linear relationship between intracellular FA-DCCDs and rhodamine 6G within a certain concentration range indicates that there is a quantitative relationship between the concentration of FA-DCCDs and rhodamine 6G and their luminescence intensity at a specified wavelength, and on this basis, the fluorescence intensity range of FA-DCCDs is within the linear range of rhodamine 6G, which can ensure that the quantification within the concentration range of rhodamine 6G can cover the quantitative range of FA-DCCDs.

[0224] 4.3. Quantitative detection:

[0225] Hela cell line was used as the culture object. After the Hela cell line was cultured in a 96-well plate for 36 hours, 10 mg / mL FA-DCCD solution was intermittently administered and cell uptake was performed at four experimental time points: 3 hours, 6 hours, 9 hours, and 12 hours. Terminate the culture. The culture medium and carbon dot mixed culture medium were aspirated and washed 3 times with PBS (pH 7.30) to wash away the residual culture medium and excess FA-DCCD solution outside the cells. 50 μL DMSO was added to each well for cell lysis to obtain a cell solution containing FA-DCCD, and 50 μL diluent: sodium chloride (0.9%) was added to obtain a test solution, wherein the concentration of FA-DCCDs in the test sample (cell lysate) is C0, and the range of C0 is 0.4988-7.980 μg / mL. The fluorescence intensity was measured and detected using an ELISA reader at an excitation wavelength of 460 nm and a wavelength channel of 560 nm to obtain the fluorescence intensity F of the test sample;

[0226] Substitute the measured fluorescence intensity F of the sample to be tested into the linear fitting equation of the rhodamine reference substance: y = 12.996x-2.9095, take y = F, and calculate the value of the concentration C' = x;

[0227] The carbon quantum dot content C in the sample to be tested is calculated according to the following formula: C%=(C' / C0)*100%.

[0228] Example 3: Establishment of a quantitative detection method for DCCDs in liver and kidney tissues in vivo

[0229] 1. Solution preparation

[0230] 1.1 Preparation of reference stock solution

[0231] Accurately weigh 0.1003 g of cresyl purple, add sodium chloride solution (0.9%) to dissolve, transfer the solution to a 100 mL volumetric flask, and dilute to obtain a 1.003 mg / mL cresyl purple reference solution. Set aside.

[0232] 1.2 Preparation of DCCDs stock solution

[0233] Accurately weigh 0.1011 g DCCDs, add sodium chloride solution (0.9%) to dissolve, transfer the solution to a 100 mL volumetric flask, and adjust the volume to obtain a 1.011 mg / mL DCCDs solution. Set aside.

[0234] 2. Effects of tissue fluid on DCCDs

[0235] The liver and kidney tissues were prepared into 10% biological tissue fluid, centrifuged at 13000r / min for 10min, and filtered with a 0.22nm microporous filter membrane to obtain the liver and kidney tissue supernatant.

[0236] Take 500 μL of DCCDs solution, add 500 μL of liver tissue fluid, mix well, and scan the DCCDs-liver tissue fluid fluorescence emission spectrum at an excitation wavelength of 570 nm.

[0237] Take 500 μL of DCCDs solution, add 500 μL of renal tissue fluid, mix well, and scan the DCCDs-renal tissue fluid fluorescence emission spectrum at an excitation wavelength of 570 nm.

[0238] The carbon dot solution, liver tissue fluid, and kidney tissue fluid were scanned to investigate the effects of liver and kidney tissue fluid on DCCDs.

[0239] Fig.10 AC showed that there was background interference between liver and kidney tissue fluids on DCCDs. At the same time, the effects of liver tissue fluid and kidney tissue fluid on DCCDs were different, so they needed to be quantitatively analyzed separately.

[0240] 3. Establishment of Quantitative Method for DCCDs in Liver Tissue Fluid

[0241] 3.1 Determination method

[0242] The liver tissue was prepared into 10% biological tissue fluid with sodium chloride (0.9%), centrifuged at 13000r / min for 10min, and filtered with a 0.22μm microporous filter to obtain liver tissue supernatant. The liver tissue supernatant was mixed with each carbon dot solution to obtain the test solution. 100μL of each test solution was added to a 96-cell plate and fluorescence was measured using an ELISA instrument with an excitation wavelength of 570nm and an emission wavelength of 634nm.

[0243] 3.2 Methodological validation

[0244] 3.2.1 Linearity and range

[0245] 3.2.1.1 Investigation of the linear relationship of DCCDs in liver tissue fluid

[0246] Take an appropriate amount of DCCDs stock solution and prepare 1.250, 2.490, 4.980, 9.970, 14.99, and 19.94 μg / mL DCCDs solutions with sodium chloride solution (0.9%). Perform the determination according to the method under "3.1 Determination Method".

[0247] Depend on Fig.11 A shows that DCCDs in liver tissue fluid has a good linear relationship in the range of 1.250-19.94 μg / mL (y=1.0193x+0.4106, R2=0.9992).

[0248] 3.2.1.2 Investigation of the linear relationship of cresyl violet in liver tissue fluid

[0249] Take the stock solution of cresyl violet reference substance and prepare 0.3906, 0.7812, 1.562, 3.125, 5.010, 6.250 μg / mL cresyl violet reference substance solutions with sodium chloride solution (0.9%). Perform the determination according to the method under "3.1 Determination Method".

[0250] Depend on Fig.11 B shows that cresyl violet in liver tissue fluid has a good linear relationship in the range of 0.3906-6.250 μg / mL (y=3.1678x-0.532, R2=0.9980).

[0251] In summary, DCCDs and cresyl violet in liver tissue fluid each have a good linear relationship within a certain concentration range, indicating that there is a quantitative relationship between the concentration of DCCDs and cresyl violet and their luminescence intensity at a specified wavelength, and on this basis, the fluorescence intensity range of DCCDs is within the linear range of cresyl violet, which can ensure that the quantification within the concentration range of cresyl violet can cover the quantitative range of DCCDs.

[0252] 3.3 Content determination

[0253] A 10 mg / mL DCCDs solution was prepared with a sodium chloride (0.9%) solution and filtered through a 0.22 μm microporous filter membrane. The filtrate was used as an injection solution. Normal Kunming mice were used as experimental subjects. 100 μL of the filtrate was injected into the body by tail vein injection. Two parallel groups were set up in the experiment. The mice were dissected 20, 30, 40, 50, and 60 minutes after administration, and their livers and kidneys were taken respectively. The tissue was processed according to "3.1 Determination Method" to obtain the supernatant, wherein the concentration of DCCDs in the sample to be tested (supernatant) is C0, and the range of C0 is 1.250-19.94 μg / mL. In the supernatant, fluorescence measurement was performed to obtain the fluorescence intensity F.

[0254] Substitute the measured fluorescence intensity F of the sample to be tested into the linear fitting equation of the cresyl violet reference substance: y = 3.1678x-0.532, take y = F, and calculate the value of the concentration C' = x;

[0255] (5) The carbon quantum dot content C in the sample to be tested is calculated according to the following formula: C%=(C' / C0)*100%.

[0256] 4. Establishment of Quantitative Method for DCCDs in Renal Tissue Fluid

[0257] 4.1 Determination method

[0258] The kidney tissue was prepared into 10% biological tissue fluid with sodium chloride (0.9%), centrifuged at 13000r / min for 10min, and filtered with a 0.22μm microporous filter membrane to obtain the kidney tissue supernatant. The kidney tissue supernatant was mixed with each carbon dot solution to obtain the test solution. 100μL of each test solution was added to a 96-cell plate and fluorescence was measured using an enzyme marker, with an excitation wavelength of 570nm and an emission wavelength of 634nm.

[0259] 4.2 Methodological validation

[0260] 4.2.1 Linearity and range

[0261] 4.2.1.1 Investigation of the linear relationship of DCCDs in renal tissue fluid

[0262] Take an appropriate amount of DCCDs stock solution and prepare 0.6231, 1.246, 2.492, 4.985, 7.500, and 9.970 μg / mL DCCDs solutions with sodium chloride solution (0.9%). Perform the determination according to the method under "4.1 Determination Method".

[0263] Depend on Fig.12 A shows that DCCDs in renal tissue fluid has a good linear relationship in the range of 0.6231 to 9.970 μg / mL (y = 1.9374x-0.0693, R 2=0.999).

[0264] 4.2.1.2 Investigation of the linear relationship of cresyl violet in renal tissue fluid

[0265] Take an appropriate amount of cresyl violet reference stock solution and prepare cresyl violet reference solutions of 0.1900, 0.3900, 0.7800, 1.560, 2.480, and 3.120 μg / mL respectively with sodium chloride solution (0.9%). Perform the determination according to the method under "4.1 Determination Method".

[0266] Depend on Fig.12 B shows that the cresol violet in liver tissue fluid has a good linear relationship in the range of 0.1900 to 3.120 μg / mL (y = 5.1277x-0.031, R 2 =0.9956).

[0267] In summary, DCCDs and cresyl violet in renal tissue fluid each have a good linear relationship within a certain concentration range, indicating that there is a quantitative relationship between the concentration of DCCDs and cresyl violet and their luminescence intensity at a specified wavelength, and on this basis, the fluorescence intensity range of DCCDs is within the linear range of cresyl violet, which can ensure that the quantification within the concentration range of cresyl violet can cover the quantitative range of DCCDs.

[0268] 4.3 Content determination

[0269] A 10 mg / mL DCCDs solution was prepared with a sodium chloride (0.9%) solution, and filtered through a 0.22 μm microporous filter membrane, and the filtrate was used as an injection solution. Normal Kunming mice were used as experimental subjects, and 100 μL of the filtrate was injected into the body by tail vein injection. The experiment set up two parallel groups. The mice were dissected 20, 30, 40, 50, and 60 minutes after administration, and their livers and kidneys were taken respectively. The tissue was processed according to "3.1 Determination Method" to obtain the supernatant, wherein the concentration of DCCDs in the sample to be tested (supernatant) is C0, and the range of C0 is 0.6231-9.970 μg / mL. In the supernatant, fluorescence measurement was performed to obtain the fluorescence intensity F.

[0270] Substitute the measured fluorescence intensity F of the sample to be tested into the linear fitting equation of the cresyl violet reference substance: y = 5.1277x-0.031, take y = F, and calculate the value of the concentration C' = x;

[0271] (5) The carbon quantum dot content C in the sample to be tested is calculated according to the following formula: C%=(C' / C0)*100%.

[0272] The beneficial effects of the method of the present invention are demonstrated by experimental examples below.

[0273] Experimental Example 1: Verification of the in vitro quantitative detection method of the present invention

[0274] 1. Validation of DCCDs quantitative method:

[0275] 1. Instrument precision

[0276] To test the stability of the instrument, the same DCCDs test solution was taken and measured according to the method under "Measurement method 2.1 of Example 1" for 6 consecutive measurements. The RSD of the fluorescence intensity of the 6 measurements was 1.46%. The results showed that the instrument used in this experiment has good precision.

[0277] 2. Repeatability

[0278] Accurately measure the appropriate amount of DCCDs stock solution, prepare low (8.000μg / mL), medium (9.980μg / mL), and high (11.97μg / mL) concentrations of DCCDs solutions, and prepare 3 samples of each concentration. Determine according to the method under "2.1 Determination Method". The results show that the average content of low, medium, and high concentrations of DCCDs is 34.31%, and the RSD is 2.34%. The experimental results show that this method has good repeatability.

[0279] 3. Intermediate precision

[0280] Accurately measure the appropriate amount of DCCDs stock solution (1.280mg / mL) to prepare DCCDs solutions with low (8.000μg / mL), medium (9.980μg / mL), and high (11.97μg / mL) concentrations. Three samples of each concentration were measured by two people using different instruments and at different times according to the method under "2.1 Determination Method". The results showed that the intermediate precision RSDs of the low, medium, and high experimental concentrations were 1.78%, 2.03%, and 2.06%, respectively. The experimental results show that this method has good intermediate precision.

[0281] 4. Sample stability

[0282] Take the same DCCD test solution, set six experimental time points of 0, 2, 4, 8, 12, and 24 hours, and perform content determination according to the method under "2.1 Determination Method". The RSD of the sample content determination at the six time points was 2.29%, indicating that the DCCD sample solution placed at room temperature for 24 hours has good stability.

[0283] 5. Recovery rate

[0284] Preparation of reference solution: Accurately measure an appropriate amount of cresyl violet reference stock solution and prepare a 3.40 μg / mL cresyl violet reference solution with ultrapure water.

[0285] Preparation of DCCDs solution: Accurately measure an appropriate amount of DCCDs stock solution (1.280 mg / mL) to prepare DCCDs solutions with low (8.000 μg / mL), medium (9.980 μg / mL), and high (11.97 μg / mL) concentrations, with 3 samples of each concentration.

[0286] Accurately pipette 1 mL of cresyl violet reference solution and 1 mL of low, medium and high concentration DCCDs solution and mix them, set up 3 portions for each concentration. Perform the determination according to "2.1 Determination Method". Results The average recovery rate of low, medium and high concentration DCCDs was 100.4%, and the RSD was 1.91%.

[0287] The above results confirm that the DCCDs samples detected by the in vitro quantitative detection method of DCCDs of the present invention have good stability, and the detection method of the present invention has good repeatability, high intermediate precision and high recovery rate.

[0288] 2. Validation of FA-DCCDs quantitative method:

[0289] 1. Instrument precision

[0290] To test the stability of the instrument, the same FA-DCCDs test solution was taken and measured according to the method under "Determination method in part 3.1 of Example 1" for 6 consecutive measurements. The RSD of the fluorescence intensity of the 6 measurements was 1.81%. The results showed that the instrument used in this experiment has good precision.

[0291] 2. Repeatability

[0292] Accurately measure the appropriate amount of FA-DCCDs stock solution, prepare three concentrations of FA-DCCDs solutions, low (12.78μg / mL), medium (16.04μg / mL), and high (19.30μg / mL), and prepare three samples of each concentration. Determine according to the method under "3.1 Determination Method". The results show that the average content of FA-DCCDs at low, medium, and high concentrations is 22.87%, and the RSD is 2.61%. The experimental results show that this method has good repeatability.

[0293] 3. Intermediate precision

[0294] Accurately measure the appropriate amount of FA-DCCDs stock solution, prepare three concentrations of FA-DCCDs solutions, low (12.78μg / mL), medium (16.04μg / mL), and high (19.30μg / mL), and prepare three samples of each concentration. According to the method under "3.1 Determination Method", two experimenters performed the determination with different instruments at different times. The results showed that the intermediate precision RSDs of low, medium, and high concentrations were 1.70%, 1.44%, and 1.82%, respectively. The experimental results show that this method has good intermediate precision.

[0295] 4. Sample stability

[0296] Take the same FA-DCCD test solution and perform content determination according to the method under "3.1 Determination Method" by setting 6 experimental time points, namely 0, 2, 4, 8, 12, and 24 hours. The RSD of the result is 1.67%, indicating that the content of the FA-DCCD sample solution placed at room temperature for 24 hours has good stability.

[0297] 5. Recovery rate

[0298] Preparation of reference solution: Take an appropriate amount of Rhodamine 6G reference stock solution and prepare a 3.59 μg / mL Rhodamine 6G reference solution with ultrapure water.

[0299] Preparation of FA-DCCDs solution: Accurately measure an appropriate amount of FA-DCCDs stock solution and use ultrapure water to prepare three FA-DCCDs solutions of low (12.78 μg / mL), medium (16.04 μg / mL), and high (19.30 μg / mL) concentrations.

[0300] Accurately pipette 1mL of rhodamine 6G reference solution and 1mL of low, medium and high concentration FA-DCCDs solution and mix them evenly. Set up 3 portions for each concentration. Perform the determination according to the method under "3.2 Determination Method". The average recovery rate of FA-DCCDs at low, medium and high concentrations was 101.2%, and the RSD was 2.05%. The method recovery rate was good.

[0301] The FA-DCCDs sample detected by the in vitro quantitative detection method of FA-DCCDs of the present invention has good stability, and the detection method of the present invention has good repeatability, high intermediate precision and high recovery rate.

[0302] 3. Validation of ACUCDs quantitative method:

[0303] 1. Instrument precision

[0304] To test the stability of the instrument, the same ACUCDs test solution was taken and measured according to the method under "Example 1 Part 4.1 Determination Method" for 6 consecutive times. The RSD of the fluorescence intensity of the 6 measurements was 1.88%. The results showed that the instrument used in this experiment has good precision.

[0305] 2. Repeatability

[0306] Accurately measure the appropriate amount of ACUCDs stock solution, prepare three concentrations of ACUCDs solution, low (6.292μg / mL), medium (7.865μg / mL), and high (9.438μg / mL), and prepare 3 samples of each concentration. Determine according to the method under "4.1 Determination Method". The results show that the content of ACUCDs in low, medium, and high concentrations is 21.54%, and the RSD is 1.86%. The results show that the method has good repeatability.

[0307] 3. Intermediate precision

[0308] Accurately measure the appropriate amount of ACUCDs stock solution, prepare three concentrations of ACUCDs solution, low (6.292μg / mL), medium (7.865μg / mL), and high (9.438μg / mL), and prepare three samples of each concentration. According to the method under "4.1 Determination Method", two people perform the determination in different instruments and at different times. The results show that the intermediate precision RSD of low, medium, and high concentrations are 2.76%, 2.80%, and 2.54%, respectively. The experimental results show that this method has good intermediate precision.

[0309] 4. Sample stability

[0310] Take the same ACUCDs test solution and perform the content determination according to the method under "4.1 Determination Method" with 6 experimental time points of 0, 2, 4, 8, 12, and 24 hours. The RSD of the sample stability experiment was 2.68%. The experimental results show that the ACUCD sample solution placed at room temperature for 24 hours has good stability.

[0311] 5. Recovery rate

[0312] Preparation of reference solution: Take an appropriate amount of Rhodamine 6G reference stock solution and prepare a 1.658 μg / mL Rhodamine 6G reference solution with ultrapure water.

[0313] Preparation of ACUCD solution: Accurately measure an appropriate amount of ACUCDs stock solution, prepare ACUCDs solutions of low (6.292μg / mL), medium (7.865μg / mL), and high (9.438μg / mL) concentrations, prepare 3 samples of each concentration, and measure according to the method under "4.1 Measurement method".

[0314] Accurately pipette 1mL of rhodamine 6G reference solution and 1mL of low, medium and high concentration ACUCD solutions respectively and mix them evenly. Set up 3 portions for each concentration and shake them well. Perform the determination according to the method under "4.1 Determination Method". The results show that the average recovery rate of low, medium and high concentration ACUCD is 99.94%, and the RSD is 2.20%.

[0315] The above results confirm that the ACUCDs samples detected by the in vitro quantitative detection method of ACUCDs of the present invention have good stability, and the detection method of the present invention has good repeatability, high intermediate precision and high recovery rate.

[0316] 4. Validation of FA-ACUCDs quantitative method:

[0317] 1. Instrument precision

[0318] To test the stability of the instrument, the same FA-ACUCDs test solution was taken and measured according to the method under "Determination method in part 5.1 of Example 1" for 6 consecutive times. The RSD of the fluorescence intensity of the 6 times was 1.81%. The results showed that the instrument used in this experiment has good precision.

[0319] 2. Repeatability

[0320] Accurately measure the appropriate amount of FA-ACUCDs stock solution, prepare low (48.13μg / mL), medium (60.16μg / mL), and high (72.19μg / mL) concentrations of FA-ACUCDs solutions, and prepare 3 samples of each concentration. Determine according to the method under "5.1 Determination Method". The results show that the content of FA-ACUCDs in the three concentrations of low, medium, and high is 18.18%, and the RSD is 2.25%. The experimental results show that this method has good repeatability.

[0321] 3. Intermediate precision

[0322] Accurately measure the appropriate amount of FA-ACUCDs stock solution, prepare low (48.13μg / mL), medium (60.16μg / mL), and high (72.19μg / mL) concentrations of FA-ACUCDs solutions, and prepare 3 samples of each concentration. According to the method under "5.1 Determination Method", two people perform the determination in different instruments and at different times. The results show that the intermediate precision RSDs of low, medium, and high concentrations of FA-ACUCDs are 2.36%, 2.30%, and 2.06%, respectively. The experimental results show that this method has good intermediate precision.

[0323] 4. Sample stability

[0324] Take the same FA-ACUCDs test solution and perform the assay according to the method in "5.1 Assay Method" at 0, 2, 4, 8, 12, and 24 hours. The RSD of the sample assay was 2.19%. The experimental results show that the ACUCD sample solution placed at room temperature for 24 hours can still ensure accuracy.

[0325] 5. Recovery rate

[0326] Preparation of reference solution: Take an appropriate amount of Rhodamine 6G reference stock solution and prepare a 11.06 μg / mL Rhodamine 6G reference solution with ultrapure water.

[0327] Preparation of FA-ACUCD solution: Accurately measure an appropriate amount of FA-ACUCD stock solution and use ultrapure water to prepare low (48.13 μg / mL), medium (60.16 μg / mL), and high (72.19 μg / mL) concentrations of FA-ACUCDs solutions.

[0328] Accurately pipette 1mL of rhodamine 6G reference solution and 1mL of low, medium and high concentration FA-ACUCD solution and mix them evenly. Set up 3 copies for each concentration. Perform the determination according to the method under "5.1 Determination Method". The results show that the average recovery rate of FA-ACUCD at low, medium and high concentrations is 101.7%. The average RSD is 2.56%.

[0329] The above results confirm that the FA-ACUCDs samples detected by the in vitro quantitative detection method of FA-ACUCDs of the present invention have good stability, and the detection method of the present invention has good repeatability, high intermediate precision and high recovery rate.

[0330] 5. Verification of UCPG-Fe quantitative method:

[0331] 1. Instrument precision

[0332] To test the stability of the instrument, the same UCPG-Fe test solution was taken and measured according to the method under "Determination method in part 6.1 of Example 1" for 6 consecutive measurements. The RSD of the fluorescence intensity of the 6 measurements was 1.89%. The results showed that the instrument used in this experiment has good precision.

[0333] 2. Repeatability

[0334] Accurately measure an appropriate amount of UCPG-Fe stock solution, prepare low (4.992μg / mL), medium (6.240μg / mL), and high (7.488μg / mL) UCPG-Fe solutions, set 3 samples for each concentration, and measure according to the method under "6.1 Determination Method". The results show that the content of UCPG-Fe at low, medium, and high concentrations is 26.48%, and the RSD is 2.61%. The experimental results show that this method has good repeatability.

[0335] 3. Intermediate precision

[0336] Accurately measure the appropriate amount of UCPG-Fe stock solution, prepare three concentrations of UCPG-Fe solution, low (4.992μg / mL), medium (6.240μg / mL), and high (7.488μg / mL), set 3 samples for each concentration, and measure them by two people using different instruments and at different times according to the method under "6.1 Determination Method". The RSD of the intermediate precision of low, medium, and high concentrations of UCPG-Fe were 2.11%, 2.47%, and 2.11%, respectively. The experimental results show that this method has good intermediate precision.

[0337] 4. Sample stability

[0338] Take the same UCPG-Fe test solution, set 6 experimental time points of 0, 2, 4, 8, 12, and 24h, and perform content determination according to the method under "6.1 Determination Method". The RSD of the sample stability experiment was 2.02%. The experimental results show that the accuracy of the content determination of the UCPG-Fe sample solution after being placed at room temperature for 24h can still be guaranteed.

[0339] 5. Recovery rate

[0340] Preparation of reference solution: Take an appropriate amount of Rhodamine 6G reference stock solution and prepare a 1.641 μg / mL Rhodamine 6G reference solution with ultrapure water.

[0341] Preparation of UCPG-Fe solution: Accurately measure an appropriate amount of UCPG-Fe stock solution and use ultrapure water to prepare three concentrations of UCPG-Fe solutions: low (4.992 μg / mL), medium (6.240 μg / mL), and high (7.488 μg / mL).

[0342] Accurately pipette 1mL of Rhodamine 6G reference solution and 1mL of low, medium and high concentration UCPG-Fe solution and mix them evenly. Set up 3 portions for each concentration. Perform the determination according to the method under "6.1 Determination Method". The experimental results show that the average recovery rate of low, medium and high concentration UCPG-Fe solutions is 99.61%. The RSD is 2.33%.

[0343] The above results confirm that the UCPG-Fe sample detected by the in vitro quantitative detection method of UCPG-Fe of the present invention has good stability, and the detection method of the present invention has good repeatability, high intermediate precision and high recovery rate.

[0344] 6. Validation of FA-UCPG-Fe quantitative method:

[0345] 1. Instrument precision

[0346] To test the stability of the instrument, the same FA-UCPG-Fe test solution was taken and measured according to the method under "Determination method in part 7.1 of Example 1" for 6 consecutive measurements. The RSD of fluorescence intensity in 6 measurements was 2.60%. The results showed that the instrument used in this experiment has good precision.

[0347] 2. Repeatability

[0348] Accurately measure the appropriate amount of FA-UCPG-Fe stock solution, prepare low (1.920μg / mL), medium (2.400μg / mL), and high concentration (2.880μg / mL) FA-UCPG-Fe solutions, set up 3 copies of each concentration sample, and determine according to the method under "7.1 Determination Method". The results show that the average content of UCPG-Fe at low, medium, and high concentrations is 23.47%, and the RSD is 2.04%. The experimental results show that the method has good repeatability.

[0349] 3. Intermediate precision

[0350] Accurately measure the appropriate amount of FA-UCPG-Fe stock solution, prepare low (1.920μg / mL), medium (2.400μg / mL), and high concentration (2.880μg / mL) FA-UCPG-Fe solutions, set up 3 copies of each concentration sample, and measure them by two people using different instruments and at different times according to the method under "7.1 Determination Method". The RSD of the intermediate precision of the low, medium, and high concentration FA-UCPG-Fe solutions was 1.92%, 1.42%, and 1.52%, respectively. The experimental results show that this method has good intermediate precision.

[0351] 4. Sample stability

[0352] Take the same FA-UCPG-Fe test solution, set 6 experimental time points of 0, 2, 4, 8, 12, and 24h, and determine the sample content according to the method under "7.1 Determination Method". The RSD of the sample content determination result is 1.69%. The experimental results show that the accuracy of the content determination of the FA-UCPG-Fe sample solution placed at room temperature for 24h can be guaranteed.

[0353] 5. Recovery rate

[0354] Preparation of reference solution: Take an appropriate amount of Rhodamine 6G reference stock solution and prepare it into 0.5612 μg / mL Rhodamine 6G reference solution with ultrapure water.

[0355] Preparation of FA-ACUCD solution: Accurately measure an appropriate amount of FA-UCPG-Fe stock solution and use ultrapure water to prepare FA-ACUCD solutions of low, medium and high concentrations of 1.920 mg / mL, 2.400 mg / mL and 2.880 mg / mL.

[0356] Accurately pipette 1mL of rhodamine 6G reference solution and 1mL of low, medium and high concentration FA-UCPG-Fe solution and mix them evenly. Set up 3 portions for each concentration. Perform the determination according to the method under "7.1 Determination Method". The experimental results show that the average recovery rate of low, medium and high concentrations of FA-UCPG-Fe is 99.86%. The average RSD is 2.12%.

[0357] The above results confirm that the FA-UCPG-Fe samples detected by the in vitro quantitative detection method of FA-UCPG-Fe of the present invention have good stability, and the detection method of the present invention has good repeatability, high intermediate precision and high recovery rate.

[0358] Experimental Example 2: Verification of the Intracellular Quantitative Detection Method of DCCDs and FA-DCCDs of the Present Invention

[0359] 1. Validation of the DCCDs intracellular quantitative detection method

[0360] 1. Instrument precision

[0361] To test the stability of the instrument, the same DCCDs test solution was taken and measured according to the method under "Determination method in part 3.1.2 of Example 2" for 6 consecutive measurements. The RSD of the fluorescence intensity detected 6 times was 2.96%. The results showed that the instrument used in this experiment has good precision.

[0362] 2. Intraday precision

[0363] Accurately measure the DCCDs stock solution (with cresyl violet as the control, the labeled content is 34.40%, 0.3474 mg / mL), prepare three solutions with low, medium and high concentrations, add 96-well plates with 50 μL of cell fluid, and obtain three test solutions with low (0.2316 μg / mL), medium (0.2895 μg / mL) and high (0.3474 μg / mL) concentrations. Each concentration sample has 3 copies, and the determination is carried out according to the method under "3.1.2 Determination Method of Example 2". The results show that the intra-day precision RSD of low, medium and high concentrations of DCCDs are 3.61%, 4.67% and 3.44%, respectively. The experimental results show that the intra-day precision of this method is good.

[0364] 3. Daytime precision

[0365] Accurately measure the DCCDs stock solution (with cresyl violet as the control, the labeled content is 34.40%, 0.3474 mg / mL), prepare three solutions with low, medium and high concentrations, add 96-well plates with 50 μL of cell fluid, and obtain three test solutions with low (0.2316 μg / mL), medium (0.2895 μg / mL) and high (0.3474 μg / mL) concentrations. Three samples of each concentration are measured by two people in different instruments and at different times according to the method under "3.1.2 Determination Method of Example 2". The results show that the day-to-day precision RSD of low, medium and high concentrations of DCCDs in cells are 4.15%, 4.15% and 3.41% respectively. The experimental results show that the method has good day-to-day precision.

[0366] 4. Sample stability

[0367] Take the same DCCD test solution, set 6 experimental time points of 0, 2, 4, 8, 12, and 24 hours, and perform the determination according to the method under "3.1.2 Determination Method of Example 2". The RSD of the sample content determination is 2.14%. The results show that the DCCD sample solution placed at room temperature for 24 hours has good stability.

[0368] 5. Recovery rate

[0369] Accurately measure an appropriate amount of DCCDs stock solution (the labeled content is 34.40% and 0.3474 mg / mL with cresyl violet as the control), and prepare 10 mL of each solution with three concentrations of low, medium and high content, and 3 samples of each concentration. Take 50 μL of each sample and add it to a 96-well plate with 50 μL of cell fluid to obtain three concentrations of test solution: low (0.2316 μg / mL), medium (0.2895 μg / mL), and high (0.3474 μg / mL). According to the method under "3.1.2 Determination Method of Example 2", the determination was carried out. The experimental results showed that the average recovery rate of DCCDs at low, medium and high concentrations was 102.5%, and the RSD was 3.28%.

[0370] The above results confirm that the DCCDs samples detected by the intracellular quantitative detection method of DCCDs of the present invention have good stability, the detection method of the present invention has good repeatability, high intra-day and inter-day precision and high recovery rate.

[0371] 2. Validation of the FA-DCCDs intracellular quantitative detection method

[0372] 1. Instrument precision

[0373] In order to test the stability of the instrument, the same FA-DCCDs test solution was taken and measured 6 times in succession according to the determination method under "4.1.2 Determination method". The RSD of fluorescence intensity in 6 determinations was 3.84%. The results showed that the instrument used in this experiment has good precision.

[0374] 2. Intraday precision

[0375] Accurately measure the appropriate amount of FA-DCCDs stock solution (with rhodamine 6G as the reference, the labeled content is 22.92%, 0.2285 mg / mL), prepare three solutions with low, medium and high concentrations, add 96-well plates with 50 μL of cell fluid, and obtain three test solutions with low (0.7334 μg / mL), medium (0.9168 μg / mL) and high (1.100 μg / mL) concentrations. Set 3 samples for each concentration, and determine according to the method under "4.1.2 Determination Method". Results The intra-day precision RSDs of the low, medium and high concentrations of FA-DCCDs solutions were 2.04%, 2.77% and 3.64%, respectively. The experimental results show that the intra-day precision of this method is good.

[0376] 3. Daytime precision

[0377] Accurately measure the DCCDs stock solution (with rhodamine 6G as the reference, the labeled content is 22.92%, 0.2285 mg / mL), prepare three solutions with low, medium and high concentrations, add 50 μL of cell solution to a 96-well plate, and obtain three test solutions with low (0.7334 μg / mL), medium (0.9168 μg / mL) and high (1.100 μg / mL) concentrations. Set 3 samples for each concentration, and set 3 samples for each concentration. According to the method under "4.1.2 Determination Method", two people perform the determination in different instruments and at different times. The results show that the inter-day precision RSDs of low, medium and high concentrations are 2.29%, 2.46% and 1.94% respectively. The experimental results show that this method has good inter-day precision.

[0378] 4. Sample stability

[0379] Take the same FA-DCCD test solution, set 6 experimental time points of 0, 2, 4, 8, 12, and 24h, and perform the determination according to the method under "4.1.2 Determination Method". The RSD of the sample content is 2.64%. The results show that the FA-DCCD sample solution placed at room temperature for 24h has good stability.

[0380] 5. Recovery rate

[0381] Accurately measure an appropriate amount of FA-DCCDs stock solution (with rhodamine 6G as the reference, the labeled content is 22.92%, 0.2285 mg / mL), and prepare 10 mL of three solutions with low, medium and high concentrations, respectively, and 3 samples of each concentration. Take 50 μL of each sample and add it to a 96-well plate with 50 μL of cell fluid to obtain three test solutions with low (0.7334 μg / mL), medium (0.9168 μg / mL), and high (1.100 μg / mL) concentrations. According to the method under "4.1.2 Determination Method", the determination was carried out. The results showed that the average recovery rate of the low, medium and high concentrations of FA-DCCDs solutions was 100.9%, and the RSD was 3.87%, showing good accuracy.

[0382] The above results confirm that the FA-DCCDs samples detected by the intracellular quantitative detection method of FA-DCCDs of the present invention have good stability, the detection method of the present invention has good repeatability, high intra-day and inter-day precision and high recovery rate.

[0383] The above results show that within a certain concentration range, the candidate carbon dots and their FA conjugates have a good linear relationship, and the cell fluid environment does not interfere with the fluorescence spectrum of the carbon dots, reflecting the anti-bleaching property of the carbon dots. At the same time, it can avoid the complicated pretreatment process of biological samples, and detection can be achieved by directly lysing the cells, indicating that the method of the present invention has the potential to achieve rapid detection.

[0384] Experimental Example 3: Verification of the quantitative detection method of DCCDs in tissues of the present invention

[0385] 1. Recovery and precision

[0386] Liver tissue fluid: Accurately measure an appropriate amount of DCCDs stock solution (with cresyl violet as the reference, the labeled content is 34.40%, 0.3474 mg / mL), and prepare DCCDs sample solutions with four concentrations of quantitative lower limit, low, medium and high with sodium chloride (0.9%) solution, with 5 samples of each concentration. Take 100 μL of liver tissue fluid, 100 μL of each sample, mix well, and obtain four concentrations of test sample solutions of quantitative lower limit (0.4300 μg / mL), low (1.720 μg / mL), medium (3.440 μg / mL), and high (5.160 μg / mL), and perform fluorescence determination according to "3.1 Determination Method".

[0387] Test results: The recovery rate of DCCDs in liver tissue fluid was between 86.61% and 108.8%, the intra-day precision RSD was between 4.83% and 8.53%, and the inter-day precision RSD was between 0.26% and 2.80%. The results showed that this method has good precision.

[0388] Renal tissue fluid: Accurately measure an appropriate amount of DCCDs stock solution (with cresyl violet as the reference, the labeled content is 34.40%, 0.3474 mg / mL), and prepare DCCDs sample solutions with four concentrations of quantitative lower limit, low, medium and high with sodium chloride (0.9%) solution, with 5 samples of each concentration. Take 100 μL of renal tissue fluid, 100 μL of each sample, mix evenly, and obtain four concentrations of test sample solutions of quantitative lower limit (0.2334 μg / mL), low (0.4736 μg / mL), medium (1.915 μg / mL) and high (2.876 μg / mL), and perform fluorescence determination according to "4.1 Determination Method".

[0389] Test results: The recovery rate of DCCDs in renal tissue fluid was between 89.83% and 112.9%, the RSD of intra-day precision was between 3.49% and 8.76%, and the RSD of inter-day precision was between 0.95% and 2.64%.

[0390] 2. Stability

[0391] The DCCDs stock solution was mixed evenly with liver tissue fluid and kidney tissue fluid, respectively, and placed at room temperature and -20℃ freezing conditions for a period of time, and fluorescence detection was performed to calculate the average concentration and judge the average accuracy. It was found that the DCCDs tissue fluid sample could be stably placed at room temperature for more than 8 hours, and the average accuracy was still close to 100%. After being placed at -20℃ for 30 days, the average accuracy was still as high as 100%; and after repeated freezing and thawing at -20℃ for 3 times, the detection content was still stable. This shows that the DCCDs tissue fluid sample is very stable and has great potential in actual clinical application.

[0392] The above results indicate that within a certain concentration range, the linear range of the candidate carbon dots in tissue fluid is good, the tissue fluid does not affect the detection of carbon dots, and the sensitivity and intra-day and inter-day precision meet the detection requirements.

[0393] In summary, the present invention provides a method for quantitatively detecting the content of carbon dots in vitro, in cells, and in tissues, which has high accuracy, good repeatability, high precision, and high recovery rate. It breaks through the bottleneck that there is no reference substance for carbon dots and it is difficult to conduct quantitative detection, and provides a quantitative research method for the establishment of carbon dot quality standards and in-depth research in biological diagnosis and treatment.

Claims

1. A quantitative detection method for fluorescent carbon quantum dots, characterized in that: The steps include: (1) preparing a sample solution with a concentration of C0 using a diluent, detecting the fluorescence intensity, and obtaining the fluorescence intensity F of the sample; (2) Prepare a stock solution of the reference substance with the diluent. Dilute the stock solution with the diluent to obtain at least 5 reference substance solutions with gradient concentrations of C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C22, C12, C13, C14, C15, C16, C17, C18, C29, C21, C21, C30, C19 n , detect the fluorescence intensity, and obtain the fluorescence intensity F1, F2…F corresponding to the gradient concentration of the reference solution n , n is an integer greater than or equal to 5; (3) Using the concentrations of the reference solution C1, C2, ... C n and the corresponding fluorescence intensities F1, F2…F n Draw a standard curve and obtain the fitting equation of the fluorescence intensity-concentration of the reference substance by linear fitting; (4) Substituting the fluorescence intensity F of the sample to be tested measured in step (1) into the fitting equation obtained in step (3) to calculate the concentration C'; (5) Calculate the carbon quantum dot content C in the sample to be tested according to the following formula: C% = (C' / C0)*100%; Wherein, the reference substance is cresyl violet or rhodamine; The fluorescent carbon quantum dots are DCCDs, FA-DCCDs, ACUCDs, FA-ACUCDs, UCPG-Fe or FA-UCPG-Fe; The DCCDs are carbon quantum dots prepared from citric acid, urea, phosphoric acid and N,N-dimethylformamide as raw materials, and the FA-DCCDs are tumor-targeting carbon quantum dots formed by chemical bonding of DCCDs and folic acid; The ACUCDs are carbon quantum dots prepared from ammonium citrate and urea as raw materials, and the FA-ACUCDs are tumor-targeting carbon quantum dots formed by chemical bonding of ACUCDs and folic acid; The UCPG-Fe is a carbon quantum dot prepared from citric acid, urea, PEG400 and iron salt as raw materials, and the FA-UCPG-Fe is a tumor-targeting carbon quantum dot formed by chemically bonding UCPG-Fe and folic acid.

2. The detection method according to claim 1, characterized in that The sample to be tested is a solid mixture or tissue fluid containing fluorescent carbon quantum dots, or a lysate obtained by lysing cells that have taken up fluorescent carbon quantum dots.

3. The detection method according to claim 2, characterized in that The tissue fluid is liver tissue fluid or kidney tissue fluid.

4. The detection method according to claim 1, characterized in that The diluent is ultrapure water or sodium chloride solution.

5. The detection method according to claim 4, characterized in that: The concentration of the sodium chloride solution is 0.9%wt.

6. The detection method according to claim 1, characterized in that The fluorescence intensity is detected by using an ELISA instrument to detect the fluorescence intensity at an emission wavelength λm under an excitation wavelength λ; The emission wavelength λm is the maximum emission wavelength of the fluorescent carbon quantum dots, and the excitation wavelength λ is the excitation wavelength corresponding to the fluorescent carbon quantum dots when the maximum emission wavelength is obtained.

7. The detection method according to claim 1, characterized in that The fluorescent carbon quantum dots are DCCDs, and the reference substance is cresyl violet; the fluorescence intensity is detected by detecting the fluorescence intensity at an emission wavelength of 634 nm under an excitation wavelength of 570 nm.

8. The detection method according to claim 1, characterized in that The fluorescent carbon quantum dots are FA-DCCDs, and the reference substance is rhodamine; the fluorescence intensity is the fluorescence intensity at an emission wavelength of 560 nm detected under an excitation wavelength of 460 nm.

9. The detection method according to claim 1, characterized in that: The fluorescent carbon quantum dots are ACUCDs, and the reference substance is rhodamine; the fluorescence intensity is detected by detecting the fluorescence intensity at an emission wavelength of 570 nm under an excitation wavelength of 490 nm.

10. The detection method according to claim 1, characterized in that: The carbon quantum dots are FA-ACUCDs, the reference substance is rhodamine; the fluorescence intensity is the fluorescence intensity at an emission wavelength of 534 nm detected under an excitation wavelength of 420 nm.

11. The detection method according to claim 1, characterized in that: The carbon quantum dots are UCPG-Fe, and the reference substance is rhodamine; the fluorescence intensity is detected by detecting the fluorescence intensity at an emission wavelength of 555 nm under an excitation wavelength of 480 nm; Or, the carbon quantum dots are FA-UCPG-Fe, and the reference substance is rhodamine; the fluorescence intensity is the fluorescence intensity at an emission wavelength of 545 nm detected under an excitation wavelength of 480 nm.

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