A fluorescent probe material for drug screening and a method for drug screening

Abstract

The application relates to the frontiers of nanomaterials and drug screening fields, and discloses a fluorescent probe material for drug screening and a drug screening method. The fluorescent probe material realizes screening of anticancer drug efficacy of a to-be-detected drug by detecting the concentration of an NAD+ (nicotinamide adenine dinucleotide) active marker of lactate dehydrogenase, the fluorescent probe material comprises an A agent and a B agent, the A agent is a covalent organic framework material loaded with carbon dots with reducibility, and is denoted as COF@CDs, and the B agent is a transition metal hydride; the transition metal hydride can make NAD+ hydrogenated and reduced into NADH in the presence of the COF@CDs; the product after hydrogen loss of the transition metal hydride can be combined with the COF@CDs, and the COF@CDs can emit fluorescence under excitation light, and the fluorescence is quenched.

Description

Technical Field

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

[0002] Antitumor drugs are a class of medications used to treat cancer. Simply put, they include chemotherapy drugs and biological agents. In recent years, advancements 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; and the research and development of antitumor drugs has entered a new era.

[0003] Cancer cells typically generate energy by enhancing anaerobic glycolysis of glucose, specifically by using lactate dehydrogenase to convert glucose into lactate. Therefore, the activity of lactate dehydrogenase in cancer cells is significantly higher than in normal cells. Nicotinamide adenine dinucleotide (NAD+) is a coenzyme factor for lactate dehydrogenase during anaerobic glycolysis. In cancer cells where the metabolic rate is too rapid, leading to lactate accumulation, the concentration of NAD+ is much higher than in normal cells. Therefore, the activity of lactate dehydrogenase can be reflected by measuring the concentration of NAD+. By detecting the activity of lactate dehydrogenase, the inhibitory effect of anticancer drugs on lactate dehydrogenase activity can be determined. Thus, the detection of NAD+ concentration can be used for screening the efficacy of anticancer drugs.

[0004] Carbon dots possess excellent biocompatibility and superior optical properties, making them widely used as probe materials in ion detection, nucleic acid detection, and other substance detection fields. Examples include the carbon dot fluorescent probe for glutathione detection disclosed in CN110554012A and its preparation method, and CN110607173B, which discloses CN110607173B, etc.

[0005] The inventor's previous research achievement, "CN118258799B Nanofluorescent Sensor for Drug Screening, its Preparation Method and Drug Screening Method," successfully applied carbon dots to the detection of NAD+. It also provides a feasible drug screening strategy. The nanofluorescent sensor is a ratiometric sensor based on fluorescence intensity detection. It can characterize the NAD+ concentration by the ratio of blue fluorescence to yellow fluorescence, thereby determining the efficacy of the analyte drug in inhibiting lactate dehydrogenase activity. The ratiometric sensor design reduces the influence of background fluorescence. The principle is that when NAD+ is present in the system, the phosphate group on NAD+ reacts with Fe... 3+ It exhibits stronger coordination, thereby competitively acquiring Fe from carbon dots. 3+Fe@NAD+ coordination compounds are formed. After centrifugation, the coordination compounds separate from the carbon dots, 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] The scheme achieved some results, but it also has some shortcomings or areas for further improvement: In this scheme, CDs first react with Fe... 3+ The fluorescence is quenched by binding, and then Fe is obtained competitively from CDs by NAD+. 3+ This allows for the recovery of CDs fluorescence, and the detection of NAD+ concentration is achieved through the intensity of the recovered CDs fluorescence. However, in competitive Fe... 3+ During the process, NAD+ binds to Fe 3+ Competition intensity, Fe 3+ Whether or not the CDs are sufficiently detached can affect the recovery of CDs fluorescence, which can ultimately affect the detection of NAD+ concentration.

[0007] Therefore, it is now 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 and a drug screening method for drug screening, which addresses the shortcomings of the prior art.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a fluorescent probe material for drug screening, which realizes the screening of the anticancer efficacy of the 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 reduce NAD+ to NADH by hydrogenation. 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.

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

[0011] S1. Carbon dots with reducing properties, denoted as CDs, are prepared by hydrothermal reaction of glucose, L-cysteine, and 3-methyl-4-isopropylphenol.

[0012] S2. Using 3,3',3”-(1,3,5-benzyltriyltri-2,1-ethyndiyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and dihydrazine terephthalate as raw materials, a covalent organic framework material, denoted as COF, was synthesized by a solvothermal method.

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

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

[0015] S1. Preparation of reducing carbon dot 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 reaction vessel and reacted under an inert gas atmosphere and heating. After the reaction was completed, the product was filtered, and the filtrate was dialyzed through a dialysis bag. The dialysate was collected, freeze-dried, and carbon dots were obtained, denoted as CDs.

[0017] S2. Preparation of covalent organic framework (COF) materials:

[0018] 3,3',3”-(1,3,5-benzyltriyltri-2,1-ethynyldiyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and dihydrazine terephthalate were added to a mixed solvent consisting of mesitylene and 1,4-dioxane, and sonicated. Then, acetic acid solution was added, and the resulting mixture was degassed by freezing. The mixture was then heated to react. After the reaction was completed, the mixture was centrifuged, washed, dried, and ground to obtain a covalent organic framework material, denoted as COF.

[0019] S3. Take COF and add it to ethanol, then disperse it by ultrasonication to obtain dispersion 1; take CDs and add them to ethanol, then disperse them by ultrasonication to obtain dispersion 2; add dispersion 2 to dispersion 1, shake on a shaker, and then heat until the solvent evaporates to dryness to obtain a covalent organic framework material loaded with carbon dots, denoted as COF@CDs, i.e. Agent A.

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

[0021] S1. Preparation of reducing carbon dot CDs:

[0022] Take 0.4-1.75g ​​glucose, 0.181-0.724g L-cysteine, and 0.23-0.9g 3-methyl-4-isopropylphenol and add them to a mixture of 50-2000mL ethanol and 25-100mL deionized water. Disperse the mixture by sonication for 5-30min. Transfer the resulting mixture to a polytetrafluoroethylene-lined reactor and react it at 160-200℃ under N2 atmosphere for 5-18h. Cool to room temperature and filter the product through a 0.22μm filter membrane. Dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 800-1200Da for 12-48h. Collect the dialysate in the dialysis bag, freeze-dry it, and obtain carbon dots, denoted as CDs.

[0023] S2. Preparation of covalent organic framework (COF) materials:

[0024] 0.1-0.4 mmol of 3,3',3”-(1,3,5-benzyltriyltri-2,1-ethynyldiyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and 0.15-0.6 mmol of dihydrazine terephthalate were added to 7.5-30 mL of a mixed solvent consisting of mesitylene and 1,4-dioxane in a 1:1 volume ratio. The mixture was sonicated for 2-10 min, and then 0.75-3 mL of acetic acid solution with a concentration of 1.5-6 mol / L was added. After mixing thoroughly, the resulting mixture was frozen in liquid nitrogen at 77 K and degassed by 2-5 freeze-thaw cycles. After degassed, the mixture was sealed and heated at 110-130 °C for 48-84 hours. After the reaction was completed, the solid product was collected by centrifugation, washed with tetrahydrofuran, dried under vacuum at 60-90 °C to constant weight, and ground to obtain a covalent organic framework material, denoted as COF.

[0025] S3. Take 0.25-1g COF and add it to 50-200mL ethanol, and sonicate for 15-60min to obtain dispersion 1; take 0.05-0.2g CDs and add it to 25-100mL ethanol, and sonicate 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℃ until the solvent evaporates to dryness to obtain a covalent organic framework material loaded with carbon dots, denoted as COF@CDs, i.e. Agent A.

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

[0027] S1. Preparation of reducing carbon dot CDs:

[0028] 0.85 g glucose, 0.362 g L-cysteine, and 0.45 g 3-methyl-4-isopropylphenol were added to a mixture of 100 mL ethanol and 50 mL deionized water and ultrasonically dispersed for 15 min. The resulting mixture was transferred to a polytetrafluoroethylene-lined reactor and reacted at 180 °C for 9 h under N2 atmosphere. After cooling to room temperature, the product was filtered through a 0.22 μm filter membrane. The filtrate was dialyzed through a dialysis bag with a molecular weight cutoff of 1000 Da for 24 h. The dialysate in the dialysis bag was collected, freeze-dried, and carbon dots were obtained, denoted as CDs.

[0029] S2. Preparation of covalent organic framework (COF) materials:

[0030] 0.2 mmol of 3,3',3”-(1,3,5-benzyltriyltri-2,1-ethynyldiyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and 0.3 mmol of dihydrazine terephthalate were added to 15 mL of a mixed solvent of mesitylene and 1,4-dioxane in a volume ratio of 1:1. The mixture was sonicated for 5 min, and then 1.5 mL of 3 mol / L acetic acid solution was added. After mixing thoroughly, 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 was completed, the solid product was collected by centrifugation, washed with tetrahydrofuran, dried under vacuum at 70 °C to constant weight, and ground to obtain a covalent organic framework material, denoted as COF.

[0031] S3. Take 0.5g COF and add it to 100mL ethanol, sonicate for 30min to obtain dispersion 1; take 0.1g CDs and add it to 50mL ethanol, sonicate for 30min to obtain dispersion 2; add dispersion 2 to dispersion 1 under stirring, shake on a shaker for 4h, and then heat at 90℃ until the solvent evaporates to dryness to obtain a covalent organic framework material loaded with carbon dots, denoted as COF@CDs, i.e. Agent A.

[0032] Preferably, agent B is selected from iron-hydrogen complex 4, iron-hydrogen complex 5, or iron-hydrogen complex 6 reported in the literature “Wang Yangyang. Study on catalytic properties of trimethylphosphine-supported benzene-selenophenol-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 described above to screen the anticancer efficacy of a drug to be tested. The method includes the following steps:

[0034] 1) Disperse agent A in deionized water to prepare a dispersion of agent A;

[0035] 2) Add the test drug to the dispersion of agent A, and culture cancer cells in the resulting mixture;

[0036] 3) Disperse agent B in dimethyl sulfoxide to prepare a dispersion of agent B;

[0037] 4) Add agent B dispersion to the product of step 2), sonicate, stir and react, centrifuge, discard the centrifuged liquid, redisperse the obtained product in dimethyl sulfoxide and place it under 365nm excitation light, detect the fluorescence intensity at 539nm and 620nm, and record them as F539 and F620 respectively, and calculate the value K of F539 / F620.

[0038] When K T1 ≤K≤K T2The reliability of the test results is determined by the initial assessment. Then, based on a pre-established standard curve characterizing the relationship between F620 and NAD+ concentration, the NAD+ concentration is analyzed using the F620 value. Finally, the anticancer efficacy of the test drug is determined based on the NAD+ concentration. A higher F620 value indicates a lower NAD+ concentration and better anticancer efficacy of the test drug. T1 and K T2 For a pre-set threshold;

[0039] When K > K T2 or K < K T1 If necessary, repeat steps 1)-4) to re-screen the drug for the current test drug.

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

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

[0042] 2) Add the test drug to the dispersion of agent A, and control the concentration of the test drug to be 0.2-50 μg / mL. Culture cancer cells in the resulting mixture at 37°C for 1-12 h.

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

[0044] 4) Add agent B dispersion to the product of step 2), sonicate for 10-30 min, stir at room temperature for 0.5-2 h, centrifuge, discard the centrifuged liquid, redisperse the obtained product in dimethyl sulfoxide and place it under 365 nm excitation light, detect the fluorescence intensity at 539 nm and 620 nm, and record them as F539 and F620 respectively, and calculate the value K of F539 / F620;

[0045] When K T1 ≤K≤K T2 The reliability of the test results is determined by the initial assessment. Then, based on the pre-established standard curve f1 characterizing the relationship between F620 value and NAD+ concentration, the NAD+ concentration is analyzed using the F620 value. Finally, the anticancer efficacy of the test drug is determined based on the NAD+ concentration. A higher F620 value indicates a lower NAD+ concentration and better anticancer efficacy of the test drug. T1 and K T2 For a pre-set threshold;

[0046] When K > K T2 or K < K T1 If necessary, repeat steps 1)-4) to re-screen the drug for the current test drug.

[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 min to prepare a dispersion of agent A with a concentration of 0.1-1 mg / mL;

[0050] 2) Add different concentrations of NAD+ to the dispersion of agent A, stir evenly, and prepare a series of standard working solutions containing the same concentration of agent A but different concentrations of NAD+. The NAD+ concentration range of this series of standard working solutions is 0-10 μmol / L.

[0051] 3) Add agent B to dimethyl sulfoxide and ultrasonically disperse for 15-60 min 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 min, stir at room temperature for 0.5-2 h, centrifuge, discard the centrifuged liquid, redisperse the obtained product in dimethyl sulfoxide and place it under 365 nm excitation light, detect the fluorescence intensity at 620 nm, and record it as F620; use the measured fluorescence intensity as the y-axis and the corresponding NAD+ concentration as the x-axis to perform curve fitting to obtain the standard curve f1.

[0053] The preparation process of the fluorescent probe material of the present invention and its mechanism for screening anticancer drugs:

[0054] I. Preparation process of Agent A

[0055] 1. Firstly, this invention uses glucose, L-cysteine, and 3-methyl-4-isopropylphenol as precursors to synthesize a carbon dot via a one-pot hydrothermal method. This carbon dot inherits the reducing properties of L-cysteine ​​and 3-methyl-4-isopropylphenol well, and emits bright red fluorescence at 620 nm under 365 nm excitation light. The surface of this carbon dot is rich in functional groups such as amino, hydroxyl, carboxyl, and thiol groups. These functional groups can interact strongly with transition metal ions, and the fluorescence of the carbon dot is quenched due to excited-state electron transfer and energy transfer.

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

[0057] 3. Then, using a covalent organic framework material as a carrier, a large number of carbon dots were uniformly loaded onto it, ultimately yielding Agent A: COF@CDs. COF has good stability and biocompatibility, as well as a large specific surface area and tunable optical properties. Loading with COF can improve the dispersibility of CDs.

[0058] COFs, as biocompatible nanocarriers with strong adsorption capacity, can easily enter or adsorb onto cancer cells after being loaded with CDs, thanks 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 PharmacalResearch,2025,48(3):183-211.DOI:10.1007 / s12272-025-01536-2.);When screening drugs, the A-agent dispersion and the test drug are cultured together with cancer cells. After the culture is completed, the B-agent dispersion is added. After the reaction, the mixture is centrifuged and the centrifuged liquid is discarded. Then the fluorescence intensity of the product is detected. At this time, COF@CDs can enter or adsorb onto cancer cells and are thus well preserved, which can reduce the loss during the centrifugation process and thus better ensure the accuracy of the detection results.

[0059] On the other hand, the combination of CDs and COF can modulate optical properties and enhance luminescence intensity. Specifically, the prepared COF@CDs have bimodal emission characteristics. Under 365nm excitation light, they can emit fluorescence at both 539nm and 620nm, and the fluorescence intensity of both emission peaks is enhanced.

[0060] The main principles of carbon dots enhancing COF fluorescence include: (1) Carbon dots can promote electron transfer under photoexcitation, especially in composite systems, their surface functional groups can form effective interactions with COF. This interaction can optimize charge separation efficiency, prolong excited state lifetime, and thus enhance fluorescence emission intensity (Liu Cui. Study on the structure, fluorescence properties and luminescence mechanism of carbon dots [J]. [2025-05-16].); (2) The nanoscale size and abundant surface functional groups of carbon dots make it easy to form a stable covalent composite structure with COF. This type of composite structure can effectively suppress the fluorescence quenching of COF and regulate the fluorescence emission wavelength and intensity through surface modification.

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

[0062] II. Mechanism of Anticancer Drug Screening Achieved by Combining Agent A and Agent B

[0063] Nicotinamide adenine dinucleotide (NAD+) is a coenzyme factor for lactate dehydrogenase during anaerobic glycolysis of glucose. In cancer cells where rapid metabolism leads to lactate accumulation, the concentration of NAD+ is significantly higher than in normal cells. Therefore, the activity of lactate dehydrogenase can be reflected by measuring the concentration of NAD+. By detecting the activity of lactate dehydrogenase, 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. It can transfer negative hydrogen to NAD+, realizing the hydrogenation reduction of NAD+. NAD+ is reduced to NADH. After losing hydrogen, Agent B transforms into the ionic state M+ (Zhang Fanjun. Study on the reduction of NAD+-like organic cations by metal iron complexes and the catalytic hydrocracking reaction of pyridine borosilicate [D]. Shandong University, 2017.), exposing the metal coordination bond. It can combine with COF and CDs in COF@CDs, thereby quenching the dual fluorescence of COF@CDs.

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

[0066] Furthermore, in this invention, the fluorescence intensity at 620 nm in COF@CDs is significantly enhanced. Using this as a quantitative signal to analyze NAD+ concentration can improve sensitivity and accuracy. The fluorescence at 539 nm can complement the fluorescence at 620 nm to assess the reliability of the fluorescence detection results. Specifically, the fluorescence intensities at 539 nm and 620 nm originate from the COF and CDs in COF@CDs, respectively, and are only related to the properties of COF@CDs themselves (mainly their preparation method and the ratio of COF to CDs). Therefore, for the same COF@CDs, the ratio K (K = F539 / F620) of the fluorescence intensities at 539 nm and 620 nm will remain essentially constant. For example, in some preferred embodiments, K fluctuates around 0.4. Therefore, using K as an internal reference can determine the reliability of the fluorescence test results: K fluctuating within an acceptable range around 0.4 indicates that the fluorescence test results are reliable; otherwise, the fluorescence test results are unreliable. In this way, the fluorescence test results can be assessed first, thereby ensuring the reliability of the final drug screening results.

[0067] The beneficial effects of this invention are:

[0068] (1) This invention provides a novel material that can be used for drug screening. It 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. In the presence of COF@CDs, Agent B can reduce NAD+ to NADH by hydrogenation. The product of Agent B after dehydrogenation can bind to COF@CDs and quench the fluorescence emitted by COF@CDs under excitation light. The concentration of NAD+ can be analyzed by the change in fluorescence intensity. Finally, the anticancer efficacy of the drug can be screened by the concentration of NAD+.

[0069] (2) In this invention, the hydrogenation reaction between agent B and NAD+ occurs in the presence of COF@CDs, and the fluorescence of COF@CDs is then quenched through the reaction products. Compared with the competitive binding of transition metals to achieve fluorescence quenching in the prior art, this invention can improve sensitivity and accuracy. Specifically, for example, in patent CN118258799B, Fe is obtained from CDs through competitive binding of NAD+. 3+ This causes CDs fluorescence recovery and competes with Fe. 3+ During the process, NAD+ binds to Fe 3+ Competition intensity, Fe 3+ Whether or not the CDs are sufficiently detached can affect the recovery of CDs fluorescence, ultimately affecting the detection of NAD+ concentration. However, in this invention, since there is no competing relationship, the fluorescence of COF@CDs is quenched by the dehydrogenation product of agent B, which avoids the above problems and improves the sensitivity and accuracy of detection.

[0070] (3) The present invention further combines the provided fluorescent probe material to construct a novel drug screening strategy: mix and culture agent A COF@CDs with the drug to be tested and cancer cells, and after the culture is completed, add agent B to detect the NAD+ concentration 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 ratio of their dual fluorescence are utilized. The ratio K of fluorescence intensity at 539 nm and 620 nm is used as an internal reference to determine the reliability of fluorescence test results, which can ensure the credibility of the final drug screening results. Using 620 nm, which has a higher fluorescence intensity, as a quantitative signal to analyze NAD+ concentration can improve the sensitivity and accuracy of the detection results. Attached Figure Description

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

[0073] Figure 2 The image shows a transmission electron microscope (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 are shown.

[0075] Figure 4 The fluorescence intensity test results are for COF@CDs at different concentrations;

[0076] Figure 5 For Fe 3+ Results of fluorescence quenching test on COF@CDs;

[0077] Figure 6 The results show the changes in fluorescence intensity at 539 nm and 620 nm with different NAD+ concentrations.

[0078] Figure 7 The standard curve f1 obtained from the fitting;

[0079] Figure 8 The results show the values ​​of F539 and F620, as well as the K value, for each working solution system at different NAD+ concentrations.

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

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

[0082] Figure 11 The results are the toxicity test results of the COF@CDs prepared in Example 1. Detailed Implementation

[0083] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0084] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence 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. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0086] In the following examples, agent B (transition metal hydride) was selected from iron-hydrogen complex 4 reported in the literature "Wang Yangyang. Study on catalytic properties of trimethylphosphine-supported benzene-selenophenol-based iron-hydrogen compounds [D]. Northwest Normal University, 2019," namely [cis-(H)(SeAr)Fe(PMe3)4], Ar=C6H5; it was prepared entirely according to the method described therein.

[0087] In the following examples, COF refers to the COF in the reference (Li Mengyao, Fluorescence Performance Regulation and Application of Chemiluminescent Covalent Organic Framework Materials [D]. Jiangxi Normal University. 2023-06-01.). BETH-TD The preparation method is shown below in detail.

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

[0089] 3,3',3”-(1,3,5-benzyltri-2,1-ethynyldiyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde, CAS No.: 705930-83-2, Brand: Alpha, purchased from Zhengzhou Huiju Chemical Co., Ltd.;

[0090] Glucose, L-cysteine, Shanghai Zhongfeng Biotechnology Co., Ltd.;

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

[0092] Dihydrazide terephthalate (dihydrazide terephthalate), CAS No. 136-64-1, Jiangsu Bost Chemical Technology Co., Ltd.

[0093] Example 1

[0094] A fluorescent probe material for drug screening is disclosed, which screens the anticancer efficacy of drugs 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 is prepared by the following method:

[0096] S1. Preparation of reducing carbon dot CDs:

[0097] 0.85 g glucose, 0.362 g L-cysteine, and 0.45 g 3-methyl-4-isopropylphenol were added to a mixture of 100 mL ethanol and 50 mL deionized water and ultrasonically dispersed for 15 min. The resulting mixture was transferred to a polytetrafluoroethylene-lined reactor and reacted at 180 °C for 9 h under N2 atmosphere. After cooling to room temperature, the product was filtered through a 0.22 μm filter membrane. The filtrate was dialyzed through a dialysis bag with a molecular weight cutoff of 1000 Da for 24 h. The dialysate in the dialysis bag was collected, freeze-dried, and carbon dots were obtained, denoted as CDs.

[0098] S2. Preparation of covalent organic framework (COF) materials:

[0099] 0.2 mmol of 3,3',3”-(1,3,5-benzyltriyltri-2,1-ethynyldiyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and 0.3 mmol of dihydrazine terephthalate were added to 15 mL of a mixed solvent consisting of mesitylene and 1,4-dioxane in a volume ratio of 1:1. The mixture was sonicated for 5 min, and then 1.5 mL of 3 mol / L acetic acid solution was added. After mixing thoroughly, 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 was completed, the solid product was collected by centrifugation, washed with tetrahydrofuran, dried under vacuum at 70 °C to constant weight, and ground to obtain a covalent organic framework material, denoted as COF.

[0100] S3. Take 0.5g COF and add it to 100mL ethanol, sonicate for 30min to obtain dispersion 1; take 0.1g CDs and add it to 50mL ethanol, sonicate for 30min to obtain dispersion 2; add dispersion 2 to dispersion 1 under stirring, shake on a shaker for 4h, and then heat at 90℃ until the solvent evaporates to dryness to obtain a covalent organic framework material loaded with carbon dots, denoted 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 catalytic properties of trimethylphosphine-supported benzene-selenophenol-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 the drug to be tested, includes the following steps:

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

[0105] 2) Add the test drug to the dispersion of agent A, and control the concentration of the test drug to 10 μg / mL. Culture cancer cells in the resulting mixture at 37°C for 2 h.

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

[0107] 4) Add agent B dispersion to the product of step 2), sonicate for 20 min, stir and react at room temperature for 1 h, centrifuge, discard the centrifuged liquid, redisperse the obtained product in dimethyl sulfoxide and place it under 365 nm excitation light, detect the fluorescence intensity at 539 nm and 620 nm, and record them as F539 and F620 respectively, and calculate the value K of F539 / F620.

[0108] When K T1 ≤K≤K T2 The reliability of the test results is determined by the initial assessment. Then, based on the pre-established standard curve f1 characterizing the relationship between F620 value and NAD+ concentration, the NAD+ concentration is analyzed using the F620 value. Finally, the anticancer efficacy of the test drug is determined based on the NAD+ concentration. A higher F620 value indicates a lower NAD+ concentration and better anticancer efficacy of the test drug. T1 and K T2 For a pre-set threshold;

[0109] When K > K T2 or K < K T1 If necessary, repeat steps 1)-4) to re-screen the drug for the current test drug.

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

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

[0112] Performance characterization:

[0113] 1. Reference Figure 1 The infrared spectra of CDs, COF, and COF@CDs prepared in Example 1 are shown; in COF, the 1620 cm⁻¹... -1 The nearby characteristic peaks originate from the C=N in the Schiff base reaction products of BTTH and TD, similar to the COF reported in the literature (Li Mengyao, Fluorescence Performance Regulation and Application of Chemiluminescent Covalent Organic Framework Materials [D]. Jiangxi Normal University. 2023-06-01.). BETH-TD Consistent. Combining the infrared spectra of CDs, COF, and COF@CDs, it can be seen that the COF@CDs after CDs were loaded onto carbon dots exhibited the 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) originated from the loaded carbon dots, which indicates the successful synthesis of CDs and COF, as well as the successful loading of CDs onto COF.

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

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

[0116] The results are as follows Figure 3 As shown, the emission peak of COF is near 539 nm, the emission peak of CDs is near 620 nm, and COF@CDs shows emission peaks at both 620 nm and 539 nm, with the intensity of the emission peaks being significantly enhanced. The fluorescence intensity at 620 nm is significantly stronger than that at 539 nm.

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

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

[0119] Test results are as follows Figure 4 As shown, within the above concentration range, the fluorescence intensity gradually increases with the increase of dispersion concentration, but the ratio K of fluorescence intensity at 539 nm and 620 nm 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 Fe2+ with different concentrations was added to each COF@CDs dispersion. 3+ The solutions (0, 1, 2, 3, 4, 5 mg / mL) were specifically Fe(NO3)3 solutions. The solution was stirred for 15 min, and then the fluorescence intensity at 539 nm and 620 nm was measured under an excitation light of 356 nm. These were recorded as F539 and F620, respectively. The K value was calculated as K = F539 / F620.

[0122] 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 539 nm and 620 nm, with Fe 3+ With the increase of amount, the fluorescence intensity at both locations gradually decreased, while the ratio K of the fluorescence intensity at 539nm and 620nm remained basically unchanged, always around 0.4.

[0123] 6. Develop the standard curve f1

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

[0125] 1) The agent A (COF@CDs) prepared in Example 1 was added to deionized water and ultrasonically dispersed for 30 min to prepare an agent A dispersion with a concentration of 1 mg / mL;

[0126] 2) Add different concentrations of NAD+ to the dispersion of agent A, stir evenly, and prepare a series of standard working solutions containing the same concentration of agent A but different concentrations of NAD+. The NAD+ concentration range of this 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 min 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 min, stir at room temperature for 1 h, centrifuge, discard the centrifuged liquid, redisperse the resulting product in dimethyl sulfoxide, and place it under 365 nm excitation light. Detect the fluorescence intensity at 539 nm and 620 nm, denoted as F539 and F620, respectively. Plot the measured fluorescence intensity F620 as the y-axis and the corresponding NAD+ concentration as the x-axis, and perform curve fitting to obtain the standard curve f1. Then calculate the K value: K = F539 / F620.

[0129] The fluorescence intensity at 539 nm and 620 nm varies with different NAD+ concentrations as shown in the following results. Figure 6 As shown, it can be seen that the fluorescence intensity at both locations gradually decreases with increasing NAD+ concentration; the fitted standard curve f1 is as follows. Figure 7 As shown, the relevance R 2 =0.9956, indicating a good linear relationship.

[0130] The values ​​of F539 and F620, as well as the K value, for each working solution system at different NAD+ concentrations are shown below. Figure 8 As shown. This result, combined with the experimental results in 4 and 5, demonstrates that the ratio K of the fluorescence intensity at 539 nm and 620 nm of COF@CDs is not affected by its concentration, and even if it is quenched (by Fe... 3+ Even if quenched (either by the product of hydrogen loss from transition metal hydrides in the NAD+ and B agent system), the ratio K remains essentially unchanged, indicating that the ratio K depends only on the intrinsic properties of COF@CDs. Therefore, this ratio K can be used as an internal standard to determine the reliability of fluorescence test results. For the COF@CDs prepared in Example 1, if the ratio K fluctuates within an allowable range of around 0.4 (specifically 0.35–0.45, i.e., K0.45), the ratio K is considered acceptable. T1=0.35, K T2 A ratio K of 0.45 indicates reliable fluorescence test results. The NAD+ concentration can be analyzed using the F620 value to determine the anticancer efficacy of the test drug. If the ratio K deviates significantly from 0.4, the fluorescence test results are unreliable. Possible causes include improper fluorescence testing procedures, problems with the fluorescence equipment, deterioration of COF@CDs during storage, uneven dispersion of the COF@CDs dispersion, or other improper procedures during the testing process. Using the ratio K as an internal reference to pre-judge the fluorescence test results ensures the reliability of the final drug screening results.

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

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

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

[0134] 2) Add different concentrations of NAD+ to the COF dispersion, stir evenly, and prepare a series of test solutions containing the same concentration of agent A but different concentrations of NAD+. The NAD+ concentration range in this series of 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 min 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 min, stir and react at room temperature for 1 h, centrifuge, discard the centrifuged liquid, redisperse the obtained product in dimethyl sulfoxide and place it under 365 nm excitation light, and detect the fluorescence intensity at 539 nm, which is recorded as F539-COF.

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

[0138] Test results are as follows Figure 9As shown, in the presence of NAD+, the fluorescence of CDs at 620 nm can be significantly quenched by agent B, and the higher the NAD+ concentration, the greater the decrease in fluorescence intensity. This indicates that in the presence of CDs, agent B can efficiently hydrogenate NAD+ to NADH. However, the fluorescence intensity of COF at 539 nm is not significantly quenched by agent B, indicating that when COF is present alone, agent B cannot efficiently achieve the hydrogenation reduction of NAD+ to NADH.

[0139] 8. Reproducibility test of COF@CDs

[0140] The COF@CDs prepared in Example 1 were dispersed in ethanol to obtain COF@CDs dispersions of different concentrations (0-1 mg / mL). The reducing power of these COF@CDs dispersions was then tested using a DPPH reagent kit (Isehisa (Jiangsu Lianyungang) Biotechnology Co., Ltd., specification 100T / 96S). The DPPH reagent exhibits strong absorption at 515 nm. Upon addition of an antioxidant (reducing agent), a decolorization reaction occurs, resulting in a decrease in absorbance at 515 nm. Within a certain range, the change in absorbance is directly proportional to the reducing power. Therefore, the degree of decrease in absorbance at 515 nm can reflect the reducing power of COF@CDs.

[0141] Test results are as follows Figure 10 As shown, COF@CDs exhibits good reducing properties. Within the range of 0-1 mg / mL, the absorbance at 515 nm gradually decreases with increasing concentration, 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 along with the test drug, it is necessary to test the biotoxicity of Agent A.

[0144] Using HeLa cells as the experimental subject, the toxicity of agent A (COF@CDs) prepared in Example 1 was detected using the MTT Cell Proliferation and Cytotoxicity Assay Kit. The x-axis represents the concentration of COF@CDs, and the y-axis represents cell viability. The test results are as follows: Figure 11 As shown, COF@CDs in the concentration range of 0.1-5 mg / mL have very low cytotoxicity.

[0145] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A fluorescent probe material for drug screening, which screens the anticancer efficacy of a drug 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 reduce NAD+ to NADH by hydrogenation. 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. Agent A is prepared by the following method: S1. Carbon dots with reducing properties, denoted as CDs, are prepared by hydrothermal reaction of glucose, L-cysteine, and 3-methyl-4-isopropylphenol. S2. Using 3,3',3''-(1,3,5-benzyltriyltri-2,1-ethynediyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and dihydrazine terephthalate as raw materials, a covalent organic framework material, denoted as COF, was synthesized by a solvothermal method. S3. Load CDs onto COF to obtain a covalent organic framework material with loaded carbon dots, denoted as COF@CDs, i.e. Agent A; The method for screening the anticancer efficacy of a drug using the aforementioned fluorescent probe material includes the following steps: 1) Disperse agent A in deionized water to prepare a dispersion of agent A; 2) Add the test drug to the dispersion of agent A, and culture cancer cells in the resulting mixture; 3) Disperse agent B in dimethyl sulfoxide to prepare a dispersion of agent B; 4) Add agent B dispersion to the product of step 2), sonicate, stir and react, centrifuge, discard the centrifuged liquid, redisperse the obtained product in dimethyl sulfoxide and place it under 365nm excitation light, detect the fluorescence intensity at 539nm and 620nm, and record them as F539 and F620 respectively, and calculate the value K of F539 / F620. When K T1 ≤K≤K T2 The reliability of the test results was determined by the time the results were assessed, and then a pre-established standard curve characterizing the relationship between F620 values ​​and NAD+ concentration was used. f 1 The NAD+ concentration was obtained by analyzing the F620 value, and the anticancer efficacy of the test drug was determined based on the NAD+ concentration; the larger the F620 value, the lower the NAD+ concentration, and the better the anticancer efficacy of the test drug; K T1 and K T2 For a pre-set threshold; When K > K T2 or K < K T1 If necessary, repeat steps 1)-4) to re-screen the drug for the current test drug.

2. The fluorescent probe material for drug screening according to claim 1, characterized in that, Agent A is prepared by the following method: S1. Preparation of reducing carbon dot 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 reaction vessel and reacted under an inert gas atmosphere and heating. After the reaction was completed, the product was filtered, and the filtrate was dialyzed through a dialysis bag. The dialysate was collected, freeze-dried, and carbon dots were obtained, denoted as CDs. S2. Preparation of covalent organic framework (COF) materials: 3,3',3''-(1,3,5-benzyltriyltri-2,1-ethynyldiyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and dihydrazine terephthalate were added to a mixed solvent consisting of mesitylene and 1,4-dioxane, and sonicated. Then, acetic acid solution was added, and the resulting mixture was degassed by freezing. The mixture was then heated to react. After the reaction was completed, the mixture was centrifuged, washed, dried, and ground to obtain a covalent organic framework material, denoted as COF. S3. Take COF and add it to ethanol, then disperse it by ultrasonication to obtain dispersion 1; take CDs and add them to ethanol, then disperse them by ultrasonication to obtain dispersion 2; add dispersion 2 to dispersion 1, shake on a shaker, and then heat until the solvent evaporates to dryness to obtain a covalent organic framework material loaded with carbon dots, 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 is prepared by the following method: S1. Preparation of reducing carbon dot CDs: Take 0.4-1.75g ​​glucose, 0.181-0.724g L-cysteine, and 0.23-0.9g 3-methyl-4-isopropylphenol and add them to a mixture of 50-2000mL ethanol and 25-100mL deionized water. Disperse the mixture by sonication for 5-30min. Transfer the resulting mixture to a polytetrafluoroethylene-lined reactor and react it at 160-200℃ under N2 atmosphere for 5-18h. Cool to room temperature and filter the product through a 0.22μm filter membrane. Dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 800-1200Da for 12-48h. Collect the dialysate in the dialysis bag, freeze-dry it, and obtain carbon dots, denoted as CDs. S2. Preparation of covalent organic framework (COF) materials: 0.1-0.4 mmol of 3,3',3''-(1,3,5-benzyltriyltri-2,1-ethynyldiyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and 0.15-0.6 mmol of dihydrazine terephthalate were added to 7.5-30 mL of a mixed solvent consisting of mesitylene and 1,4-dioxane in a 1:1 volume ratio. The mixture was sonicated for 2-10 min, and then 0.75-3 mL of acetic acid solution with a concentration of 1.5-6 mol / L was added. After mixing thoroughly, the resulting mixture was frozen in liquid nitrogen at 77 K and degassed by 2-5 freeze-thaw cycles. After degassed, the mixture was sealed and heated at 110-130°C for 48-84 hours. After the reaction was completed, the solid product was collected by centrifugation, washed with tetrahydrofuran, dried under vacuum at 60-90°C to constant weight, and ground to obtain a covalent organic framework material, denoted as COF. S3. Take 0.25-1g COF and add it to 50-200mL ethanol, and sonicate for 15-60min to obtain dispersion 1; take 0.05-0.2g CDs and add it to 25-100mL ethanol, and sonicate 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℃ until the solvent evaporates to dryness to obtain a covalent organic framework material loaded with carbon dots, denoted as COF@CDs, i.e. Agent A.

4. The fluorescent probe material for drug screening according to claim 3, characterized in that, Agent A is prepared by the following method: S1. Preparation of reducing carbon dot CDs: 0.85 g glucose, 0.362 g L-cysteine, and 0.45 g 3-methyl-4-isopropylphenol were added to a mixture of 100 mL ethanol and 50 mL deionized water and ultrasonically dispersed for 15 min. The resulting mixture was transferred to a polytetrafluoroethylene-lined reactor and reacted at 180 °C for 9 h under N2 atmosphere. After cooling to room temperature, the product was filtered through a 0.22 μm filter membrane. The filtrate was dialyzed through a dialysis bag with a molecular weight cutoff of 1000 Da for 24 h. The dialysate in the dialysis bag was collected, freeze-dried, and carbon dots were obtained, denoted as CDs. S2. Preparation of covalent organic framework (COF) materials: 0.2 mmol of 3,3',3''-(1,3,5-benzyltriyltri-2,1-ethynyldiyl)tris[5-(1,1-dimethylethyl)-6-hydroxybenzaldehyde and 0.3 mmol of dihydrazine terephthalate were added to 15 mL of a mixed solvent consisting of mesitylene and 1,4-dioxane in a volume ratio of 1:

1. The mixture was sonicated for 5 min, and then 1.5 mL of 3 mol / L acetic acid solution was added. After mixing thoroughly, 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 was completed, the solid product was collected by centrifugation, washed with tetrahydrofuran, dried under vacuum at 70°C to constant weight, and ground to obtain a covalent organic framework material, denoted as COF. S3. Take 0.5g COF and add it to 100mL ethanol, sonicate for 30min to obtain dispersion 1; take 0.1g CDs and add it to 50mL ethanol, sonicate for 30min to obtain dispersion 2; add dispersion 2 to dispersion 1 under stirring, shake on a shaker for 4h, and then heat at 90℃ until the solvent evaporates to dryness to obtain a covalent organic framework material loaded with carbon dots, denoted as COF@CDs, i.e. Agent A.

5. The fluorescent probe material for drug screening according to claim 1, characterized in that, Agent B is an iron-hydrogen complex: [cis-(H)(SeAr)Fe(PMe3)4], Ar=C6H5.

6. The fluorescent probe material for drug screening according to claim 1, characterized in that, The method for screening the anticancer efficacy of a drug using the aforementioned fluorescent probe material includes the following steps: 1) Add agent A to deionized water and ultrasonically disperse for 10-30 min to prepare a dispersion of agent A with a concentration of 0.1-1 mg / mL; 2) Add the test drug to the dispersion of agent A, and control the concentration of the test drug to be 0.2-50 μg / mL. Culture cancer cells in the resulting mixture at 37°C for 1-12 h. 3) Add agent B to dimethyl sulfoxide and ultrasonically disperse for 15-60 min to prepare a dispersion of agent B with a concentration of 0.2-5 mg / mL; 4) Add agent B dispersion to the product of step 2), sonicate for 10-30 min, stir at room temperature for 0.5-2 h, centrifuge, discard the centrifuged liquid, redisperse the obtained product in dimethyl sulfoxide and place it under 365 nm excitation light, detect the fluorescence intensity at 539 nm and 620 nm, and record them as F539 and F620 respectively, and calculate the value K of F539 / F620; When K T1 ≤K≤K T2 The reliability of the test results was determined by the time the results were assessed, and then a pre-established standard curve characterizing the relationship between F620 values ​​and NAD+ concentration was used. f 1 The NAD+ concentration was obtained by analyzing the F620 value, and the anticancer efficacy of the test drug was determined based on the NAD+ concentration; the larger the F620 value, the lower the NAD+ concentration, and the better the anticancer efficacy of the test drug; K T1 and K T2 For a pre-set threshold; When K > K T2 or K < K T1 If necessary, repeat steps 1)-4) to re-screen the drug for the current test drug.

7. The fluorescent probe material for drug screening according to claim 6, characterized in that, in, 0.30<K T1 <K T2 <0.50。 8. The fluorescent probe material for drug screening according to claim 6, characterized in that, The standard curve f 1 It is constructed using the following method: 1) Add agent A to deionized water and ultrasonically disperse for 10-30 min to prepare a dispersion of agent A with a concentration of 0.1-1 mg / mL; 2) Add different concentrations of NAD+ to the dispersion of agent A, stir evenly, and prepare a series of standard working solutions containing the same concentration of agent A but different concentrations of NAD+. The NAD+ concentration range of this series of standard working solutions is 0-10 μmol / L. 3) Add agent B to dimethyl sulfoxide and ultrasonically disperse for 15-60 min 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 min, stir at room temperature for 0.5-2 h, centrifuge, discard the centrifuged liquid, redisperse the resulting product in dimethyl sulfoxide, and place it under 365 nm excitation light. Detect the fluorescence intensity at 620 nm, denoted as F620. Plot the measured fluorescence intensity as the y-axis and the corresponding NAD+ concentration as the x-axis, and perform curve fitting to obtain the standard curve. f 1 .

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