Fluorescent probe material for drug screening and drug screening method

Abstract

The invention relates to the field of leading-edge nanometer materials and drug screening, and discloses a fluorescent probe material for drug screening and a drug screening method. According to the fluorescent probe material, the anti-cancer efficacy of a drug to be detected is screened by detecting the concentration of a lactic dehydrogenase active marker NAD +, the fluorescent probe material comprises an agent A and an agent B, the agent A is a covalent organic framework material loaded with reductive carbon dots and marked as COF-CDs, and the agent B is a transition metal hydride and marked as COF-CDs. The transition metal hydride can enable NAD < + > to be hydrogenated and reduced into NADH in the presence of COF (at) CDs, a product obtained after the transition metal hydride is dehydrogenated can be combined with the COF (at) CDs, and fluorescence emitted by the COF (at) CDs under exciting light is quenched.

Description

Technical Field

[0001] The present invention relates to the field of cutting-edge nanomaterials and drug screening, and in particular to a fluorescent probe material for drug screening and a drug screening method. Background Art

[0002] Anti-tumor drugs are a class of medications used to treat tumors. Briefly, they include chemotherapy drugs and biologics. In recent years, advances in molecular oncology and molecular pharmacology have gradually clarified the nature of tumors. The invention and application of advanced technologies such as large-scale rapid screening, combinatorial chemistry, and genetic engineering have accelerated drug development, ushering in a new era in the research and development of anti-tumor drugs.

[0003] Cancer cells typically provide themselves with energy by enhancing the anaerobic glycolysis of glucose, converting glucose into lactate through the enzyme lactate dehydrogenase. Therefore, the activity of lactate dehydrogenase in cancer cells is much higher than in normal cells. Nicotinamide adenine dinucleotide (NAD+) is a coenzyme factor for lactate dehydrogenase during the anaerobic glycolysis of glucose. In cancer cells with rapid metabolic rates, which cause lactate accumulation, the concentration of NAD+ in cancer cells is much higher than that in normal cells. Therefore, measuring the concentration of NAD+ can reflect the activity of lactate dehydrogenase. By measuring the activity of lactate dehydrogenase, the inhibitory effect of anticancer drugs on lactate dehydrogenase activity can be determined. Therefore, measuring the concentration of NAD+ can be used to screen the efficacy of anticancer drugs.

[0004] Carbon dots have good biocompatibility and excellent optical properties and are widely used as probe materials in ion detection, nucleic acid detection, and other substance detection fields. For example, CN110554012A discloses a carbon dot fluorescent probe for glutathione detection and its preparation method, and CN110607173B discloses a carbon dot fluorescent probe for glutathione detection and its preparation method.

[0005] The inventor's previous research results "CN118258799B Nanofluorescence Sensor for Drug Screening, Its Preparation Method and Drug Screening Method" successfully applied carbon dots to the detection of NAD+, and also provided a feasible drug screening strategy. The nanofluorescence sensor is a ratiometric sensor based on fluorescence intensity detection, which can characterize the NAD+ concentration by the ratio of blue fluorescence to yellow fluorescence, thereby realizing the efficacy judgment of the drug to be tested for inhibiting lactate dehydrogenase activity. The design of the ratiometric sensor can reduce the influence of background fluorescence. The principle is: when NAD+ exists in the system, the phosphate group on NAD+ reacts with Fe 3+ It has a stronger coordination effect, thus competing with the Fe on the carbon dots. 3+, forming Fe@NAD+ complex, which is separated from the carbon dots after centrifugation, thereby restoring the blue fluorescence of the carbon dots; the intensity of the restored blue fluorescence is positively correlated with the concentration of NAD+.

[0006] This scheme has achieved certain results, but there are also some shortcomings or areas for further improvement: In this scheme, CDs first react with Fe 3+ The fluorescence is quenched by binding, and then NAD+ competes for Fe from CDs. 3+ The CDs fluorescence is restored and the NAD+ concentration is detected by the CDs fluorescence recovery intensity. 3+ During the process, NAD+ binds to Fe 3+ Competition intensity, Fe 3+ Whether the cell is sufficiently separated from CDs will affect the recovery of CDs fluorescence, and ultimately affect the detection of NAD+ concentration.

[0007] Therefore, it is necessary to improve existing technologies to provide more reliable solutions or more alternatives. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a fluorescent probe material for drug screening and a drug screening method in view of the deficiencies in the above-mentioned prior art.

[0009] To solve the above technical problems, the technical solution adopted by the present invention is: a fluorescent probe material for drug screening, which screens the anti-cancer efficacy of the drug to be tested by detecting the concentration of NAD+, a lactate dehydrogenase activity marker. The fluorescent probe material includes agent A and agent B, wherein agent A is a covalent organic framework material loaded with reducing carbon dots, denoted as COF@CDs, and agent B is a transition metal hydride. In the presence of COF@CDs, the transition metal hydride can hydrogenate and reduce NAD+ to NADH. The product after the transition metal hydride loses hydrogen can bind to COF@CDs and quench the fluorescence emitted by COF@CDs under excitation light.

[0010] Preferably, Agent A is prepared by the following method:

[0011] S1, using glucose, L-cysteine, and 3-methyl-4-isopropylphenol through hydrothermal reaction to prepare reducing carbon dots, denoted as CDs;

[0012] S2, using 3,3',3"-(1,3,5-benzyltriyltri-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and terephthalic acid dihydrazine as raw materials, a covalent organic framework material was synthesized by a solvothermal method, denoted as COF;

[0013] S3. CDs are loaded onto COF to obtain a covalent organic framework material loaded with carbon dots, which is denoted as COF@CDs, i.e., agent A.

[0014] Preferably, Agent A is prepared by the following method:

[0015] S1. Preparation of reducible carbon dots CDs:

[0016] Glucose, L-cysteine, and 3-methyl-4-isopropylphenol were added to a mixture of ethanol and deionized water and dispersed by ultrasonication. The resulting mixture was transferred to a reactor and reacted under an inert gas atmosphere and heating. After the reaction, the product was filtered and the filtrate was dialyzed using a dialysis bag. The dialyzate was collected and freeze-dried to obtain carbon dots, which were recorded as CDs.

[0017] S2. Preparation of covalent organic framework material COF:

[0018] 3,3',3"-(1,3,5-benzyltriyltri-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde] and terephthalic acid dihydrazine were added to a mixed solvent consisting of mesitylene and 1,4-dioxane, and ultrasonicated. Then, an acetic acid solution was added. The resulting mixture was freeze-degassed and heated for reaction. After the reaction, the mixture was centrifuged, washed, dried, and ground to obtain a covalent organic framework material, which was recorded as COF.

[0019] S3. Take COF and add it to ethanol, and ultrasonically disperse it to obtain dispersion 1; take CDs and add it to ethanol, and ultrasonically disperse it to obtain dispersion 2; add dispersion 2 to dispersion 1, shake it on a shaker, and then heat it until the solvent is evaporated to obtain a covalent organic framework material loaded with carbon dots, which is recorded as COF@CDs, i.e., agent A.

[0020] Preferably, Agent A is prepared by the following method:

[0021] S1. Preparation of reducible carbon dots CDs:

[0022] 0.4-1.75 g of glucose, 0.181-0.724 g of L-cysteine, and 0.23-0.9 g of 3-methyl-4-isopropylphenol were added to a mixture of 50-2000 mL of ethanol and 25-100 mL of deionized water. The mixture was ultrasonically dispersed for 5-30 min. The resulting mixture was transferred to a polytetrafluoroethylene-lined reactor and reacted at 160-200°C under a nitrogen atmosphere for 5-18 h. After cooling to room temperature, the product was filtered through a 0.22 μm filter membrane, and the filtrate was dialyzed through a dialysis bag with a molecular weight cutoff of 800-1200 Da for 12-48 h. The dialyzate in the dialysis bag was collected and freeze-dried to obtain carbon dots, which were designated as CDs.

[0023] S2. Preparation of covalent organic framework material COF:

[0024] 0.1-0.4 mmol of 3,3',3"-(1,3,5-benzyltriyltris-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and 0.15-0.6 mmol of terephthalic acid dihydrazine were added to 7.5-30 mL of a mixed solvent consisting of mesitylene and 1,4-dioxane in a volume ratio of 1:1, and ultrasonicated for 2-10 minutes. Then, 0.75-3 mL of 1.5-6 mol / L acetic acid solution was added, and the mixture was mixed evenly. The resulting mixture was frozen in liquid nitrogen at 77K and degassed by 2-5 freeze-thaw cycles; after degassed, it was sealed and heated at 110-130°C for 48-84 hours. After the reaction, the solid product was collected by centrifugation and washed with tetrahydrofuran, dried in vacuo at 60-90°C to constant weight, and ground to obtain a covalent organic framework material, which was recorded as COF;

[0025] S3. Take 0.25-1g COF and add it to 50-200mL ethanol, and ultrasonically disperse it for 15-60min to obtain dispersion 1; take 0.05-0.2g CDs and add it to 25-100mL ethanol, and ultrasonically disperse it for 15-60min to obtain dispersion 2; add dispersion 2 to dispersion 1 with stirring, shake on a shaker for 2-8h, and then heat at 70-95°C until the solvent is evaporated to obtain a covalent organic framework material loaded with carbon dots, recorded as COF@CDs, i.e., agent A.

[0026] Preferably, Agent A is prepared by the following method:

[0027] S1. Preparation of reducible carbon dots CDs:

[0028] 0.85 g of glucose, 0.362 g of L-cysteine, and 0.45 g of 3-methyl-4-isopropylphenol were added to a mixture of 100 mL of ethanol and 50 mL of deionized water. The mixture was ultrasonically dispersed for 15 min and transferred to a polytetrafluoroethylene-lined reactor. The reaction was carried out at 180°C under a nitrogen atmosphere for 9 h. The mixture was cooled to room temperature and filtered through a 0.22 μm filter membrane. The filtrate was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da. The dialysate in the dialysis bag was collected and freeze-dried to obtain carbon dots, which were designated as CDs.

[0029] S2. Preparation of covalent organic framework material COF:

[0030] 0.2 mmol of 3,3',3"-(1,3,5-benzyltriyltris-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde] and 0.3 mmol of terephthalic acid dihydrazine were added to a mixed solvent consisting of 15 mL of mesitylene and 1,4-dioxane in a volume ratio of 1:1, and ultrasonicated for 5 minutes. Then, 1.5 mL of 3 mol / L acetic acid solution was added and mixed evenly. The resulting mixture was frozen in liquid nitrogen at 77 K and degassed by three freeze-thaw cycles. After degassed, the mixture was sealed and heated at 120°C for 72 hours. After the reaction, the solid product was collected by centrifugation and washed with tetrahydrofuran. It was vacuum-dried at 70°C to constant weight and ground to obtain a covalent organic framework material, which was recorded as COF.

[0031] S3. Take 0.5 g of COF and add it to 100 mL of ethanol, and ultrasonically disperse it for 30 minutes to obtain dispersion 1; take 0.1 g of CDs and add it to 50 mL of ethanol, and ultrasonically disperse it for 30 minutes to obtain dispersion 2; add dispersion 2 to dispersion 1 with stirring, shake on a shaker for 4 hours, and then heat at 90°C until the solvent is evaporated to obtain a covalent organic framework material loaded with carbon dots, which is recorded as COF@CDs, i.e., agent A.

[0032] Preferably, the agent B is selected from the iron-hydrogen complex 4 or iron-hydrogen complex 5 or iron-hydrogen complex 6 reported in the document "Wang Yangyang. Study on the catalytic properties of trimethylphosphine-supported phenylselenol-based iron-hydrogen compounds [D]. Northwest Normal University, 2019."

[0033] A second aspect of the present invention provides a drug screening method, which uses the fluorescent probe material as described above to screen the anticancer efficacy of a drug to be tested, and the method comprises the following steps:

[0034] 1) dispersing Agent A in deionized water to prepare an Agent A dispersion;

[0035] 2) adding the drug to be tested to the dispersion of Agent A, and incubating cancer cells with the resulting mixture;

[0036] 3) dispersing Agent B in dimethyl sulfoxide to prepare an Agent B dispersion;

[0037] 4) adding the dispersion of Agent B to the product of step 2), sonicating, stirring, reacting, centrifuging, discarding the centrifuge, and redispersing the resulting product in dimethyl sulfoxide and placing it under 365 nm excitation light. Detecting the fluorescence intensity at 539 nm and 620 nm, respectively, as F539 and F620, and calculating the value K of F539 / F620;

[0038] When K T1 ≤K≤K T2The test result is reliable when the F620 value is determined. Then, according to the pre-established standard curve representing the relationship between the F620 value and the NAD+ concentration, the F620 value is used to analyze the NAD+ concentration. Finally, the anticancer efficacy of the drug to be tested is determined based on the NAD+ concentration. Among them, the larger the F620 value, the lower the NAD+ concentration, and the better the anticancer efficacy of the drug to be tested. T1 and K T2 is a pre-set threshold;

[0039] When K>K T2 Or K<K T1 When the number of drug residues is less than 1%, steps 1) to 4) are repeated to re-screen the drug to be tested.

[0040] Preferably, the method comprises the following steps:

[0041] 1) Add Agent A to deionized water and ultrasonically disperse for 10-30 minutes to prepare a dispersion of Agent A with a concentration of 0.1-1 mg / mL;

[0042] 2) adding the drug to be tested to the dispersion of Agent A, controlling the concentration of the drug to be tested to be 0.2-50 μg / mL, and incubating cancer cells with the resulting mixture at 37° C. for 1-12 hours;

[0043] 3) Add Agent B to dimethyl sulfoxide and ultrasonically disperse for 15-60 minutes to prepare a dispersion of Agent B with a concentration of 0.2-5 mg / mL;

[0044] 4) Add the dispersion of Agent B to the product of step 2), sonicate for 10-30 minutes, stir and react at room temperature for 0.5-2 hours, centrifuge, discard the centrifuge, and redisperse the resulting product in dimethyl sulfoxide and place under 365 nm excitation light. Detect the fluorescence intensity at 539 nm and 620 nm, which are recorded as F539 and F620, respectively, and calculate the value K of F539 / F620;

[0045] When K T1 ≤K≤K T2 The test result is then judged to be reliable, and then the NAD+ concentration is obtained by analyzing the F620 value based on the pre-established standard curve f1 that represents the relationship between the F620 value and the NAD+ concentration. Finally, the anticancer efficacy of the drug to be tested is judged based on the NAD+ concentration. Among them, the larger the F620 value, the lower the NAD+ concentration, and the better the anticancer efficacy of the drug to be tested. T1 and K T2 is a pre-set threshold;

[0046] When K>K T2 Or K<K T1 When the number of drug residues is less than 1%, steps 1) to 4) are repeated to re-screen the drug to be tested.

[0047] Preferably, 0.30 < K T1 <K T2 <0.50.

[0048] Preferably, the standard curve f1 is constructed by the following method:

[0049] 1) Add Agent A to deionized water and ultrasonically disperse for 10-30 minutes to prepare a dispersion of Agent A with a concentration of 0.1-1 mg / mL;

[0050] 2) adding different concentrations of NAD+ to the Agent A dispersion and stirring uniformly to prepare a series of standard working solutions containing the same concentration of Agent A and different NAD+ concentrations, wherein the NAD+ concentration range of the series of standard working solutions is 0-10 μmol / L;

[0051] 3) Add Agent B to dimethyl sulfoxide and ultrasonically disperse for 15-60 minutes to prepare a dispersion of Agent B with a concentration of 0.5-5 mg / mL;

[0052] 4) Add the same volume and concentration of Agent B dispersion to each standard working solution, sonicate for 10-30 minutes, stir and react at room temperature for 0.5-2 hours, centrifuge, discard the centrifuge, redisperse the resulting product in dimethyl sulfoxide, and expose to 365 nm excitation light. Detect the fluorescence intensity at 620 nm, recorded as F620; use the measured fluorescence intensity as the y-axis and the corresponding NAD+ concentration as the x-axis for curve fitting to obtain the standard curve f1.

[0053] Preparation process of the fluorescent probe material of the present invention and its mechanism for anticancer drug screening:

[0054] 1. Preparation process of Agent A

[0055] 1. First, the present invention uses glucose, L-cysteine, and 3-methyl-4-isopropylphenol as precursors to synthesize carbon dots through a one-pot hydrothermal method. The carbon dots well inherit the reducing properties of L-cysteine ​​and 3-methyl-4-isopropylphenol and can emit bright red fluorescence at 620nm under 365nm excitation light. The surface of the carbon dots is rich in functional groups such as amino, hydroxyl, carboxyl, and thiol. These functional groups can strongly coordinate with transition metal ions, resulting in fluorescence quenching of the carbon dots due to excited-state electron transfer and energy transfer.

[0056] 2. Then, a covalent organic framework material COF was synthesized by a solvothermal method. The covalent organic framework material was prepared according to the reference literature (Li Mengyao, Fluorescence Performance Regulation and Application of Chemiluminescent Covalent Organic Framework Materials [D]. Jiangxi Normal University. 2023-06-01.), which can emit green fluorescence at 539nm under 365nm excitation light; COF has a porous structure and strong adsorption capacity, and it can also form non-fluorescent complexes through coordination with transition metal ions, resulting in fluorescence quenching.

[0057] 3. A covalent organic framework (COF) was then used as a carrier to uniformly load a large number of carbon dots, ultimately yielding Agent A: COF@CDs. COFs offer excellent stability and biocompatibility, a large specific surface area, and tunable optical properties. The COF loading enhances the dispersion of CDs.

[0058] COFs are nanocarriers with good biocompatibility and strong adsorption capacity. After loading CDs, COF@CDs can easily enter cancer cells or be adsorbed onto cancer cells due to the high permeability and retention effect (EPR effect) of tumors (Iranpour S, Abrishami A, Saljooghi A S. Covalent organic frame works in cancer theranostics: advancing biomarker detection and tumor-targeted therapy[J]. Archives of Pharmacal Research, 2025, 48(3): 183-211. DOI: 10.1007 / s12272-025-01536-2.); During drug screening, the A dispersion and the drug to be tested are incubated with cancer cells. After incubation, the B dispersion is added, the reaction is centrifuged, the centrifuge is discarded, and the fluorescence intensity of the product is detected. At this time, since COF@CDs can enter or adsorb onto cancer cells, they are well retained, which can reduce their loss during the centrifugation process, thereby better ensuring the accuracy of the test results.

[0059] On the other hand, the combination of CDs and COF can regulate optical properties and enhance luminescence intensity. Specifically, the prepared COF@CDs has a dual-peak emission characteristic. Under 365nm excitation light, it can emit fluorescence at 539nm and 620nm, and the fluorescence intensity of the two emission peaks is enhanced.

[0060] The main principles of carbon dots enhancing COF fluorescence include: (1) carbon dots can promote electron transfer under light excitation, especially in composite systems, where their surface functional groups can form effective interactions with COF, which can optimize charge separation efficiency, prolong the excited state lifetime, and thus enhance the fluorescence emission intensity (Liu Cui. Research on the structure, fluorescence properties and luminescence mechanism of carbon dots [J]. [2025-05-16].); (2) The nanometer size and rich surface functional groups of carbon dots make it easy for them to form stable covalent composite structures with COF; this type of composite structure can effectively inhibit the fluorescence quenching of COF and regulate the fluorescence emission wavelength and intensity through surface modification.

[0061] The main principles of COF-enhanced carbon dot fluorescence include: (1) COFs have a highly ordered porous structure and adjustable pore size, which can fix carbon dots through physical confinement and reduce the fluorescence quenching phenomenon caused by aggregation; (2) COFs' surface functional groups (such as amino and carboxyl groups) form hydrogen bonds or chemical bonds with carbon dot surface groups, optimizing the electronic structure through charge transfer or energy transfer. This synergistic effect can stabilize the excited state of carbon dots, extend the fluorescence lifetime and improve the luminescence efficiency; (3) COFs can promote the efficient separation and energy transfer of photogenerated carriers.

[0062] 2. Mechanism of Anticancer Drug Screening by Combining Agents A and B

[0063] Nicotinamide adenine dinucleotide (NAD+) is a coenzyme factor for lactate dehydrogenase during the anaerobic glycolysis of glucose. In cancer cells, where rapid metabolism leads to lactate accumulation, the concentration of NAD+ is much higher than in normal cells. Therefore, measuring NAD+ concentration can reflect lactate dehydrogenase activity. By measuring lactate dehydrogenase activity, the inhibitory effect of anticancer drugs on lactate dehydrogenase activity can be determined, ultimately enabling the screening of anticancer drug efficacy.

[0064] Agent B is a transition metal hydride, denoted as MH, which can transfer negative hydrogen to NAD+ to achieve hydrogenation reduction of NAD+. NAD+ is reduced to NADH. After losing hydrogen, agent B is converted into ionic M+ (Zhang Fanjun. Research on the reduction of NAD+-like organic cations by metal iron complexes and catalytic pyridine hydroboration reaction [D]. Shandong University, 2017.), exposing the metal coordination bond, which can combine with COF and CDs in COF@CDs, thereby quenching the dual fluorescence of COF@CDs.

[0065] In the present invention, agent B is selected from the iron-hydrogen complex reported in the document "Wang Yangyang. Study on the catalytic properties of trimethylphosphine-supported phenylselenol-based iron-hydrogen compounds [D]. Northwest Normal University, 2019." Agent B has catalytic reduction properties, and CDs in COF@CDs has excellent reducing properties. Agent B and COF@CDs are cleverly combined, so that when COF@CDs are present, agent B can efficiently react with NAD+, so that NAD+ is reduced to NADH. At the same time, after losing hydrogen, agent B efficiently quenches the dual fluorescence of COF@CDs, so that the dual fluorescence signal of COF@CDs can be used to detect the NAD+ concentration, thereby realizing the screening of the efficacy of anticancer drugs.

[0066] Furthermore, in the present invention, the fluorescence intensity at 620 nm in COF@CDs is significantly enhanced, and using it as a quantitative signal for analyzing NAD+ concentration can improve sensitivity and accuracy. The fluorescence at 539 nm can be used in conjunction with the fluorescence at 620 nm to determine the reliability of the fluorescence detection results. Specifically, the fluorescence intensities at 539 nm and 620 nm come from the COF and CDs in the COF@CDs, respectively, and are only related to the properties of the COF@CDs themselves (primarily their preparation method and the ratio of COF and CDs therein). Therefore, for the same COF@CDs, the ratio of the fluorescence intensities at 539 nm and 620 nm, K (K = F539 / F620), remains essentially unchanged. For example, in some preferred embodiments, K fluctuates around 0.4. Therefore, K is used as an internal reference to determine the reliability of the fluorescence test results: if K fluctuates within the allowable range around 0.4, the fluorescence test results are reliable; otherwise, the fluorescence test results are unreliable. In this way, the fluorescence test results can be pre-judged, thereby ensuring the reliability of the final drug screening results.

[0067] The beneficial effects of the present invention are:

[0068] (1) The present invention provides a new material that can be used for drug screening, which is a combined probe material, including agent A as a fluorescent emitter and agent B as a trigger for fluorescence quenching. Agent A is a covalent organic framework material loaded with reducing carbon dots, and agent B is a transition metal hydride. Agent B can hydrogenate and reduce NAD+ to NADH in the presence of COF@CDs. The product after agent B loses hydrogen can bind to COF@CDs and quench the fluorescence emitted by COF@CDs under excitation light. The concentration of NAD+ can be analyzed by changing the fluorescence intensity, and finally the anticancer efficacy screening of drugs can be achieved by the concentration of NAD+.

[0069] (2) In the present invention, the B agent reacts with NAD+ in the presence of COF@CDs to undergo a hydrogenation reaction, and then the COF@CDs fluorescence is quenched by the reaction product. Compared with the fluorescence quenching method of competitive binding of transition metals in the prior art, the present invention can improve sensitivity and accuracy. Specifically, for example, in patent CN118258799B, NAD+ is relied on to compete with CDs for Fe 3+ This restores CDs fluorescence and competes for Fe 3+ During the process, NAD+ binds to Fe 3+ Competition intensity, Fe 3+ Whether or not the COF@CDs are sufficiently separated from the CDs will affect the recovery of CDs fluorescence, ultimately affecting the detection of NAD+ concentration. However, in the present invention, since there is no competitive relationship, the COF@CDs fluorescence is quenched by the dehydrogenation product of the B agent reaction, which can avoid the above problems and thus improve the sensitivity and accuracy of detection.

[0070] (3) The present invention further combines the provided fluorescent probe material to construct a new drug screening strategy: the A-agent COF@CDs is mixed with the drug to be tested and cancer cells for cultivation. After the cultivation is completed, the NAD+ concentration is detected by adding agent B to determine the anticancer efficacy of the drug to be tested;

[0071] In this method, the dual fluorescence characteristics of COF@CDs and the fixed nature of their dual fluorescence ratio are utilized. The ratio K of the fluorescence intensities at 539nm and 620nm is used as an internal reference to judge the reliability of the fluorescence test results, which can ensure the credibility of the final drug screening results. The higher fluorescence intensity of 620nm is used as a quantitative signal to analyze NAD+ concentration, which can improve the sensitivity and accuracy of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 IR spectra of CDs, COF, and COF@CDs prepared in Example 1;

[0073] Figure 2 This is a transmission electron microscopy (TEM) image of the COF@CDs prepared in Example 1;

[0074] Figure 3 The emission spectra of CDs, COF, and COF@CDs prepared in Example 1;

[0075] Figure 4 Fluorescence intensity test results of COF@CDs with different concentrations;

[0076] Figure 5 Fe 3+ Test results of fluorescence quenching effect of COF@CDs;

[0077] Figure 6 The fluorescence intensity at 539 nm and 620 nm changes with the NAD+ concentration;

[0078] Figure 7 is the standard curve f1 obtained by fitting;

[0079] Figure 8 The results of F539, F620 and K values ​​corresponding to each working solution system at different NAD+ concentrations;

[0080] Figure 9 The results of the performance test of COF and CDs fluorescence quenching by agent B in the presence of NAD+;

[0081] Figure 10 The reducibility test results of COF@CDs prepared in Example 1;

[0082] Figure 11 These are the toxicity test results of COF@CDs prepared in Example 1. DETAILED DESCRIPTION

[0083] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0084] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0085] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Materials and reagents used in the following examples are commercially available unless otherwise specified. In the following examples, where specific conditions are not specified, the experiments were conducted under conventional conditions or those recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0086] In the following examples, agent B (transition metal hydride) is selected from the iron-hydrogen complex 4 reported in the document "Wang Yangyang. Study on the catalytic properties of trimethylphosphine-supported phenylselenol-based iron-hydrogen compounds [D]. Northwest Normal University, 2019.", namely [cis-(H)(SeAr)Fe(PMe3)4], Ar=C6H5; it was prepared in full accordance with the method thereof.

[0087] In the following examples, COF refers to the COF in the literature (Li Mengyao, Fluorescence performance adjustment and application of chemiluminescent covalent organic framework materials [D]. Jiangxi Normal University. 2023-06-01.) BETH-TD The preparation method is obtained, and the detailed steps are shown below.

[0088] In the following examples, some of the raw materials involved are from the following sources:

[0089] 3,3',3"-(1,3,5-benzyltriyltris-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde, CAS No. 705930-83-2, brand: Alpha, purchased from Zhengzhou Huiju Chemical Co., Ltd.;

[0090] Glucose and L-cysteine ​​were purchased from Shanghai Zhongfeng Biotechnology Co., Ltd.

[0091] 3-Methyl-4-isopropylphenol, Nantong Runfeng Petrochemical Co., Ltd.;

[0092] Terephthalic acid dihydrazide (terephthalic acid dihydrazide), CAS No. 136-64-1, Jiangsu Bosite Chemical Technology Co., Ltd.

[0093] Example 1

[0094] A fluorescent probe material for drug screening, which screens the anticancer efficacy of a drug to be tested by detecting the concentration of NAD+, a marker of lactate dehydrogenase activity. The fluorescent probe material includes agent A and agent B. Agent A is a covalent organic framework material loaded with reducing carbon dots, denoted as COF@CDs. Agent B is a transition metal hydride. In the presence of COF@CDs, the transition metal hydride can hydrogenate and reduce NAD+ to NADH. The product of the transition metal hydride after dehydrogenation can bind to COF@CDs and quench the fluorescence emitted by COF@CDs under excitation light.

[0095] Agent A was prepared by the following method:

[0096] S1. Preparation of reducible carbon dots CDs:

[0097] 0.85 g of glucose, 0.362 g of L-cysteine, and 0.45 g of 3-methyl-4-isopropylphenol were added to a mixture of 100 mL of ethanol and 50 mL of deionized water. The mixture was ultrasonically dispersed for 15 min and transferred to a polytetrafluoroethylene-lined reactor. The reaction was carried out at 180°C under a nitrogen atmosphere for 9 h. The mixture was cooled to room temperature and filtered through a 0.22 μm filter membrane. The filtrate was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da. The dialysate in the dialysis bag was collected and freeze-dried to obtain carbon dots, which were designated as CDs.

[0098] S2. Preparation of covalent organic framework material COF:

[0099] 0.2 mmol of 3,3',3"-(1,3,5-benzyltriyltris-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde] and 0.3 mmol of terephthalic acid dihydrazine were added to 15 mL of a mixed solvent consisting of mesitylene and 1,4-dioxane in a volume ratio of 1:1, and ultrasonicated for 5 minutes. Then, 1.5 mL of 3 mol / L acetic acid solution was added and mixed evenly. The resulting mixture was frozen in liquid nitrogen at 77 K and degassed by three freeze-thaw cycles. After degassed, the mixture was sealed and heated at 120 ° C for 72 hours. After the reaction, the solid product was collected by centrifugation and washed with tetrahydrofuran. It was vacuum-dried at 70 ° C to constant weight and ground to obtain a covalent organic framework material, which was recorded as COF.

[0100] S3. Take 0.5 g of COF and add it to 100 mL of ethanol, and ultrasonically disperse it for 30 minutes to obtain dispersion 1; take 0.1 g of CDs and add it to 50 mL of ethanol, and ultrasonically disperse it for 30 minutes to obtain dispersion 2; add dispersion 2 to dispersion 1 with stirring, shake on a shaker for 4 hours, and then heat at 90°C until the solvent is evaporated to obtain a covalent organic framework material loaded with carbon dots, which is recorded as COF@CDs, i.e., agent A.

[0101] Agent B is selected from the iron-hydrogen complex 4 reported in the literature "Wang Yangyang. Study on the catalytic properties of trimethylphosphine-supported phenylselenol-based iron-hydrogen compounds [D]. Northwest Normal University, 2019."

[0102] Example 2

[0103] A drug screening method, which uses the fluorescent probe material of Example 1 to screen the anticancer efficacy of a drug to be tested, the method comprising the following steps:

[0104] 1) Add Agent A to deionized water and ultrasonically disperse for 30 minutes to prepare a dispersion of Agent A with a concentration of 1 mg / mL;

[0105] 2) Add the drug to be tested to the dispersion of Agent A, controlling the concentration of the drug to be tested to 10 μg / mL, and incubate cancer cells with the resulting mixture at 37°C for 2 hours;

[0106] 3) Add Agent B to dimethyl sulfoxide and ultrasonically disperse for 45 minutes to prepare a dispersion of Agent B with a concentration of 2 mg / mL;

[0107] 4) Adding the dispersion of Agent B to the product of step 2), ultrasonicating for 20 minutes, stirring at room temperature for 1 hour, centrifuging, discarding the centrifuge, and redispersing the resulting product in dimethyl sulfoxide and placing it under 365 nm excitation light. Detect the fluorescence intensity at 539 nm and 620 nm, which are recorded as F539 and F620, respectively, and calculate the value K of F539 / F620;

[0108] When K T1 ≤K≤K T2 The test result is then judged to be reliable, and then the NAD+ concentration is obtained by analyzing the F620 value based on the pre-established standard curve f1 that represents the relationship between the F620 value and the NAD+ concentration. Finally, the anticancer efficacy of the drug to be tested is judged based on the NAD+ concentration. Among them, the larger the F620 value, the lower the NAD+ concentration, and the better the anticancer efficacy of the drug to be tested. T1 and K T2 is a pre-set threshold;

[0109] When K>K T2 Or K<K T1 When the number of drug residues is less than 1%, steps 1) to 4) are repeated to re-screen the drug to be tested.

[0110] The method for constructing the standard curve f1 will be described in detail later.

[0111] In this embodiment, K T1 =0.35, K T2 =0.45.

[0112] Performance characterization:

[0113] 1. Reference Figure 1 , are the infrared spectra of CDs, COF, and COF@CDs prepared in Example 1; in COF, 1620 cm -1 The characteristic peaks near the BTTH and TD are derived from the C=N in the Schiff base reaction product, which is consistent with the COF reported in the literature (Li Mengyao, Fluorescence properties regulation and application of chemiluminescent covalent organic framework materials [D]. Jiangxi Normal University. 2023-06-01.) BETH-TD Combining the infrared spectra of CDs, COF, and COF@CDs, it can be seen that after CDs are loaded onto carbon dots, COF@CDs exhibits characteristic peaks of COF, with a slight shift in the C=N peak. The characteristic peaks of -NH, -OH, -SH, C=O, and Benzene (benzene ring) originate from the loaded carbon dots, demonstrating the successful synthesis of CDs and COF, as well as the successful loading of CDs onto COF.

[0114] 2. Reference Figure 2 , is a transmission electron microscope (TEM) photograph of COF@CDs prepared in Example 1. It can be seen that a large number of CDs are uniformly loaded on the COF with a fibrous lamellar structure.

[0115] 3. Disperse CDs, COF, and COF@CDs in dimethyl sulfoxide to prepare corresponding dispersions with a concentration of 1 mg / mL, and test the emission spectra under 356 nm excitation light.

[0116] The results are as follows Figure 3 As shown in the figure, the emission peak of COF is around 539nm, the emission peak of CDs is around 620nm, and the emission peak of COF@CDs appears around both 620nm and 539nm, and the intensity of the emission peak is significantly enhanced. The fluorescence intensity at 620nm is significantly stronger than that at 539nm.

[0117] 4. Fluorescence intensity of different concentrations of COF@

[0118] The COF@CDs prepared in Example 1 were dispersed in dimethyl sulfoxide to prepare several COF@CDs dispersions of equal volume and different concentrations (0.2, 0.4, 0.6, 0.8, and 1 mg / mL). The fluorescence intensities at 539 nm and 620 nm under 356 nm excitation light were measured, denoted as F539 and F620, respectively, and the K value was calculated, K = F539 / F620.

[0119] The test results are as follows Figure 4 As shown, it can be seen that within the above concentration range, as the concentration of the dispersion increases, the fluorescence intensity gradually increases, but the ratio K of the fluorescence intensity at 539nm and 620nm remains basically unchanged, always around 0.4.

[0120] 5. Fe 3+ Fluorescence quenching effect on COF@CDs

[0121] The COF@CDs prepared in Example 1 were dispersed in dimethyl sulfoxide to prepare several 10 mL COF@CDs dispersions with a concentration of 1 mg / mL. The same volume of Fe 3+ The solution (0, 1, 2, 3, 4, 5 mg / mL), specifically Fe(NO3)3 solution, was stirred for 15 min, and then the fluorescence intensity at 539 nm and 620 nm under 356 nm excitation light was tested, recorded as F539 and F620, respectively, and the K value was calculated, K=F539 / F620.

[0122] The test results are as follows Figure 5 As shown, it can be seen that Fe 3+ It can simultaneously quench the fluorescence of COF@CDs at 539nm and 620nm. 3+ With the increase of the amount of fluorescence, the fluorescence intensities at both locations gradually decreased, while the ratio K of the fluorescence intensities at 539 nm and 620 nm remained basically unchanged, always around 0.4.

[0123] 6. Develop standard curve f1

[0124] The construction method of f1 is as follows:

[0125] 1) Add Agent A (COF@CDs) prepared in Example 1 to deionized water and ultrasonically disperse for 30 minutes to prepare a dispersion of Agent A with a concentration of 1 mg / mL;

[0126] 2) adding different concentrations of NAD+ to the Agent A dispersion and stirring uniformly to prepare a series of standard working solutions containing the same concentration of Agent A and different NAD+ concentrations, wherein the NAD+ concentration range of the series of standard working solutions is 0-10 μmol / L (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / L);

[0127] 3) Add Agent B to dimethyl sulfoxide and ultrasonically disperse for 45 minutes to prepare a dispersion of Agent B with a concentration of 2 mg / mL;

[0128] 4) Add the same volume and concentration of Agent B dispersion to each standard working solution, sonicate for 20 minutes, stir at room temperature for 1 hour, centrifuge, discard the centrifuge, redisperse the resulting product in dimethyl sulfoxide, and expose to 365 nm excitation light. Measure the fluorescence intensity at 539 nm and 620 nm, denoted as F539 and F620, respectively. Curve fitting is performed using the measured fluorescence intensity F620 as the y-axis and the corresponding NAD+ concentration as the x-axis to obtain the standard curve f1. Calculate the K value: K = F539 / F620.

[0129] The fluorescence intensity at 539nm and 620nm changes with different NAD+ concentrations as shown in the following figure: Figure 6 As shown in the figure, it can be seen that as the NAD+ concentration increases, the fluorescence intensity at both locations gradually decreases; the standard curve f1 obtained by fitting is shown in Figure 7 As shown, the correlation R 2 =0.9956, indicating a good linear relationship.

[0130] The values ​​of F539 and F620 corresponding to each working solution system at different NAD+ concentrations, as well as the results of K value are shown in the figure below. Figure 8 This result, combined with the experimental results in 4 and 5, shows that the ratio K of the fluorescence intensity at 539nm and 620nm of COF@CDs is not affected by its concentration, and even if it is quenched (by Fe 3+ The ratio K remains essentially unchanged even when the NAD+ and agent B system is quenched by the product of hydrogen loss from the transition metal hydride. This indicates that the ratio K depends only on the properties of the COF@CDs themselves. Therefore, the ratio K can be used as an internal standard to determine whether the fluorescence test results are reliable. For the COF@CDs prepared in Example 1, if the ratio K fluctuates within the allowable range of 0.4 (specifically 0.35 to 0.45, i.e., K T1=0.35, K T2 =0.45), indicating that the fluorescence test results are reliable. The NAD+ concentration can be analyzed from the F620 value to determine the anticancer efficacy of the drug under test. If the ratio K deviates significantly from 0.4, the fluorescence test results are unreliable. Possible causes include improper fluorescence test operation, problems with the fluorescence equipment, deterioration of the COF@CDs during storage, uneven dispersion of the COF@CDs dispersion, or other improper operation during the test. By using the ratio K as an internal reference indicator to pre-evaluate the fluorescence test results, the reliability of the final drug screening results can be guaranteed.

[0131] 7. Performance test of fluorescence quenching of COF and CDs by agent B in the presence of NAD+

[0132] (1) The test method for COF is:

[0133] 1) The COF prepared in Example 1 was added to deionized water and ultrasonically dispersed for 30 minutes to prepare a COF dispersion with a concentration of 1 mg / mL;

[0134] 2) adding different concentrations of NAD+ to the COF dispersion and stirring uniformly to prepare a series of test solutions containing the same concentration of Agent A and different NAD+ concentrations, wherein the NAD+ concentration range in the test solutions is 0-10 μmol / L (0, 2, 4, 6, 8, 10 μmol / L);

[0135] 3) Add Agent B to dimethyl sulfoxide and ultrasonically disperse for 45 minutes to prepare a dispersion of Agent B with a concentration of 2 mg / mL;

[0136] 4) Add the same volume and concentration of Agent B dispersion to each standard working solution, sonicate for 20 minutes, stir at room temperature for 1 hour, centrifuge, discard the centrifuge, and redisperse the resulting product in dimethyl sulfoxide (DMSO) and expose it to 365 nm excitation light. Detect the fluorescence intensity at 539 nm and record it as F539-COF.

[0137] (2) The test method of CDs is as follows: CDs prepared in Example 1 are used to replace the above COF, and the fluorescence intensity at 620 nm is detected, which is recorded as F620-CDs. The rest of the test method is the same as that of COF.

[0138] The test results are as follows Figure 9As shown, it can be seen that in the presence of NAD+, the fluorescence of CDs at 620nm can be significantly quenched by agent B, and the higher the NAD+ concentration, the more the fluorescence intensity decreases, indicating that in the presence of CDs, agent B can efficiently hydrogenate and reduce NAD+ to NADH; while the fluorescence intensity of COF at 539nm is not significantly quenched by agent B, indicating that when COF exists alone, agent B cannot efficiently achieve the hydrogenation reduction of NAD+ to NADH.

[0139] 8. Reducibility test of COF@CDs

[0140] The COF@CDs prepared in Example 1 were dispersed in ethanol to prepare COF@CDs dispersions of varying concentrations (0-1 mg / mL). The reducibility of these COF@CDs dispersions was then tested using a DPPH reagent kit (Isejiu (Lianyungang, Jiangsu) Biotechnology Co., Ltd., specifications 100T / 96S). The DPPH reagent has a strong absorption at 515 nm. Adding an antioxidant (reducing agent) causes a decolorization reaction, resulting in a decrease in absorbance at 515 nm. Within a certain range, the change in absorbance is proportional to the reducibility. Therefore, the degree of decrease in absorbance at 515 nm can reflect the reducibility of COF@CDs.

[0141] The test results are as follows Figure 10 As shown, it can be seen that COF@CDs has good reducing properties. In the range of 0-1 mg / mL, with the increase of concentration, the absorbance at 515 nm gradually decreases, indicating that its reducing ability gradually increases.

[0142] 9. Toxicity test of agent A

[0143] Since agent A (COF@CDs) needs to be co-cultured with cancer cells together with the drug to be tested, it is necessary to test the biological toxicity of agent A.

[0144] HeLa cells were used as experimental subjects, and the toxicity of agent A (COF@CDs) prepared in Example 1 was tested using the MTT Cell Proliferation and Cytotoxicity Assay Kit. The horizontal axis represents the concentration of COF@CDs, and the vertical axis represents the cell viability. The test results are shown in Figure 2. Figure 11 As shown, it can be seen that COF@CDs in the concentration range of 0.1-5 mg / mL have very low toxicity to cells.

[0145] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A fluorescent probe material for drug screening, which screens the anticancer efficacy of a drug to be tested by detecting the concentration of NAD+, a marker of lactate dehydrogenase activity, characterized in that: The fluorescent probe material includes agent A and agent B. Agent A is a covalent organic framework material loaded with reducing carbon dots, denoted as COF@CDs; agent B is a transition metal hydride. In the presence of COF@CDs, the transition metal hydride can hydrogenate and reduce NAD+ to NADH. The product after the transition metal hydride loses hydrogen can bind to COF@CDs and quench the fluorescence emitted by COF@CDs under excitation light.

2. The fluorescent probe material for drug screening according to claim 1, characterized in that Agent A was prepared by the following method: S1, using glucose, L-cysteine, and 3-methyl-4-isopropylphenol through hydrothermal reaction to prepare reducing carbon dots, denoted as CDs; S2, using 3,3',3"-(1,3,5-benzyltriyltri-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and terephthalic acid dihydrazine as raw materials, a covalent organic framework material was synthesized by a solvothermal method, denoted as COF; S3. CDs are loaded onto COF to obtain a covalent organic framework material loaded with carbon dots, which is denoted as COF@CDs, i.e., agent A.

3. The fluorescent probe material for drug screening according to claim 2, characterized in that: Agent A was prepared by the following method: S1. Preparation of reducible carbon dots CDs: Glucose, L-cysteine, and 3-methyl-4-isopropylphenol were added to a mixture of ethanol and deionized water and dispersed by ultrasonication. The resulting mixture was transferred to a reactor and reacted under an inert gas atmosphere and heating. After the reaction, the product was filtered and the filtrate was dialyzed using a dialysis bag. The dialyzate was collected and freeze-dried to obtain carbon dots, which were recorded as CDs. S2. Preparation of covalent organic framework material COF: 3,3',3"-(1,3,5-benzyltriyltri-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde] and terephthalic acid dihydrazine were added to a mixed solvent consisting of mesitylene and 1,4-dioxane, and ultrasonicated. Then, an acetic acid solution was added. The resulting mixture was freeze-degassed and heated for reaction. After the reaction, the mixture was centrifuged, washed, dried, and ground to obtain a covalent organic framework material, which was recorded as COF. S3. Take COF and add it to ethanol, and ultrasonically disperse it to obtain dispersion 1; take CDs and add it to ethanol, and ultrasonically disperse it to obtain dispersion 2; add dispersion 2 to dispersion 1, shake it on a shaker, and then heat it until the solvent is evaporated to obtain a covalent organic framework material loaded with carbon dots, which is recorded as COF@CDs, i.e., agent A.

4. The fluorescent probe material for drug screening according to claim 3, characterized in that Agent A was prepared by the following method: S1. Preparation of reducible carbon dots CDs: 0.4-1.75 g of glucose, 0.181-0.724 g of L-cysteine, and 0.23-0.9 g of 3-methyl-4-isopropylphenol were added to a mixture of 50-2000 mL of ethanol and 25-100 mL of deionized water. The mixture was ultrasonically dispersed for 5-30 min. The resulting mixture was transferred to a polytetrafluoroethylene-lined reactor and reacted at 160-200°C under a nitrogen atmosphere for 5-18 h. After cooling to room temperature, the product was filtered through a 0.22 μm filter membrane, and the filtrate was dialyzed through a dialysis bag with a molecular weight cutoff of 800-1200 Da for 12-48 h. The dialyzate in the dialysis bag was collected and freeze-dried to obtain carbon dots, which were designated as CDs. S2. Preparation of covalent organic framework material COF: 0.1-0.4 mmol of 3,3',3"-(1,3,5-benzyltriyltris-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and 0.15-0.6 mmol of terephthalic acid dihydrazine were added to 7.5-30 mL of a mixed solvent consisting of mesitylene and 1,4-dioxane in a volume ratio of 1:1, and ultrasonicated for 2-10 minutes. Then, 0.75-3 mL of 1.5-6 mol / L acetic acid solution was added, and the mixture was mixed evenly. The resulting mixture was frozen in liquid nitrogen at 77K and degassed by 2-5 freeze-thaw cycles; after degassed, it was sealed and heated at 110-130°C for 48-84 hours. After the reaction, the solid product was collected by centrifugation and washed with tetrahydrofuran, dried in vacuo at 60-90°C to constant weight, and ground to obtain a covalent organic framework material, which was recorded as COF; S3. Take 0.25-1g COF and add it to 50-200mL ethanol, and ultrasonically disperse it for 15-60min to obtain dispersion 1; take 0.05-0.2g CDs and add it to 25-100mL ethanol, and ultrasonically disperse it for 15-60min to obtain dispersion 2; add dispersion 2 to dispersion 1 with stirring, shake on a shaker for 2-8h, and then heat at 70-95°C until the solvent is evaporated to obtain a covalent organic framework material loaded with carbon dots, recorded as COF@CDs, i.e., agent A.

5. The fluorescent probe material for drug screening according to claim 4, characterized in that Agent A was prepared by the following method: S1. Preparation of reducible carbon dots CDs: 0.85 g of glucose, 0.362 g of L-cysteine, and 0.45 g of 3-methyl-4-isopropylphenol were added to a mixture of 100 mL of ethanol and 50 mL of deionized water. The mixture was ultrasonically dispersed for 15 min and transferred to a polytetrafluoroethylene-lined reactor. The reaction was carried out at 180°C under a nitrogen atmosphere for 9 h. The mixture was cooled to room temperature and filtered through a 0.22 μm filter membrane. The filtrate was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da. The dialysate in the dialysis bag was collected and freeze-dried to obtain carbon dots, which were designated as CDs. S2. Preparation of covalent organic framework material COF: 0.2 mmol of 3,3',3"-(1,3,5-benzyltriyltris-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde] and 0.3 mmol of terephthalic acid dihydrazine were added to 15 mL of a mixed solvent consisting of mesitylene and 1,4-dioxane in a volume ratio of 1:1, and ultrasonicated for 5 minutes. Then, 1.5 mL of 3 mol / L acetic acid solution was added and mixed evenly. The resulting mixture was frozen in liquid nitrogen at 77 K and degassed by three freeze-thaw cycles. After degassed, the mixture was sealed and heated at 120 ° C for 72 hours. After the reaction, the solid product was collected by centrifugation and washed with tetrahydrofuran. It was vacuum-dried at 70 ° C to constant weight and ground to obtain a covalent organic framework material, which was recorded as COF. S3. Take 0.5 g of COF and add it to 100 mL of ethanol, and ultrasonically disperse it for 30 minutes to obtain dispersion 1; take 0.1 g of CDs and add it to 50 mL of ethanol, and ultrasonically disperse it for 30 minutes to obtain dispersion 2; add dispersion 2 to dispersion 1 with stirring, shake on a shaker for 4 hours, and then heat at 90°C until the solvent is evaporated to obtain a covalent organic framework material loaded with carbon dots, which is recorded as COF@CDs, i.e., agent A.

6. The fluorescent probe material for drug screening according to claim 1, characterized in that The agent B is selected from the iron-hydrogen complex 4 or iron-hydrogen complex 5 or iron-hydrogen complex 6 reported in the document "Wang Yangyang. Study on the catalytic properties of trimethylphosphine-supported phenylselenol-based iron-hydrogen compounds [D]. Northwest Normal University, 2019." 7. A drug screening method, characterized in that: The method uses the fluorescent probe material according to any one of claims 1 to 6 to screen the anticancer efficacy of a drug to be tested, and the method comprises the following steps: 1) dispersing Agent A in deionized water to prepare an Agent A dispersion; 2) adding the drug to be tested to the dispersion of Agent A, and incubating cancer cells with the resulting mixture; 3) dispersing Agent B in dimethyl sulfoxide to prepare an Agent B dispersion; 4) adding the dispersion of Agent B to the product of step 2), sonicating, stirring, reacting, centrifuging, discarding the centrifuge, and redispersing the resulting product in dimethyl sulfoxide and placing it under 365 nm excitation light. Detecting the fluorescence intensity at 539 nm and 620 nm, respectively, as F539 and F620, and calculating the value K of F539 / F620; When K T1 ≤K≤K T2 The test result is then judged to be reliable, and then the NAD+ concentration is obtained by analyzing the F620 value based on the pre-established standard curve f1 that represents the relationship between the F620 value and the NAD+ concentration. Finally, the anticancer efficacy of the drug to be tested is judged based on the NAD+ concentration. Among them, the larger the F620 value, the lower the NAD+ concentration, and the better the anticancer efficacy of the drug to be tested. T1 and K T2 is a pre-set threshold; When K>K T2 Or K<K T1 When the number of drug residues is less than 1%, steps 1) to 4) are repeated to perform drug screening again for the drug to be tested.

8. The drug screening method according to claim 7, characterized in that The method comprises the following steps: 1) Add Agent A to deionized water and ultrasonically disperse for 10-30 minutes to prepare a dispersion of Agent A with a concentration of 0.1-1 mg / mL; 2) adding the drug to be tested to the dispersion of Agent A, controlling the concentration of the drug to be tested to be 0.2-50 μg / mL, and incubating cancer cells with the resulting mixture at 37° C. for 1-12 hours; 3) Add Agent B to dimethyl sulfoxide and ultrasonically disperse for 15-60 minutes to prepare a dispersion of Agent B with a concentration of 0.2-5 mg / mL; 4) Add the dispersion of Agent B to the product of step 2), sonicate for 10-30 minutes, stir and react at room temperature for 0.5-2 hours, centrifuge, discard the centrifuge, and redisperse the resulting product in dimethyl sulfoxide and place under 365 nm excitation light. Detect the fluorescence intensity at 539 nm and 620 nm, which are recorded as F539 and F620, respectively, and calculate the value K of F539 / F620; When K T1 ≤K≤K T2 The test result is then judged to be reliable, and then the NAD+ concentration is obtained by analyzing the F620 value based on the pre-established standard curve f1 that represents the relationship between the F620 value and the NAD+ concentration. Finally, the anticancer efficacy of the drug to be tested is judged based on the NAD+ concentration. Among them, the larger the F620 value, the lower the NAD+ concentration, and the better the anticancer efficacy of the drug to be tested. T1 and K T2 is a pre-set threshold; When K>K T2 Or K<K T1 When the number of drug residues is less than 1%, steps 1) to 4) are repeated to perform drug screening again for the drug to be tested.

9. The drug screening method according to claim 8, characterized in that in, 0.30<K T1 <K T2 <0.50。 10. The drug screening method according to claim 8, characterized in that The standard curve f1 is constructed by the following method: 1) Add Agent A to deionized water and ultrasonically disperse for 10-30 minutes to prepare a dispersion of Agent A with a concentration of 0.1-1 mg / mL; 2) adding different concentrations of NAD+ to the Agent A dispersion and stirring uniformly to prepare a series of standard working solutions containing the same concentration of Agent A and different NAD+ concentrations, wherein the NAD+ concentration range of the series of standard working solutions is 0-10 μmol / L; 3) Add Agent B to dimethyl sulfoxide and ultrasonically disperse for 15-60 minutes to prepare a dispersion of Agent B with a concentration of 0.5-5 mg / mL; 4) Add the same volume and concentration of Agent B dispersion to each standard working solution, sonicate for 10-30 minutes, stir and react at room temperature for 0.5-2 hours, centrifuge, discard the centrifuge, redisperse the resulting product in dimethyl sulfoxide, and expose to 365 nm excitation light. Detect the fluorescence intensity at 620 nm, recorded as F620; use the measured fluorescence intensity as the y-axis and the corresponding NAD+ concentration as the x-axis for curve fitting to obtain the standard curve f1.

Images