High-throughput microfluidic screening chip and screening method for anti-cancer drugs

Abstract

The application discloses a high-throughput anti-cancer drug micro-fluidic screening chip and a screening method. The screening chip comprises a chip disc body and a plurality of screening flow channel units capable of independently performing anti-cancer drug screening and arranged in a ring array around the center of the chip disc body. The screening flow channel unit comprises a water adding port, a distribution flow channel communicated with the water adding port, a waste liquid cavity and a plurality of detection flow channel units communicated between the distribution flow channel and the waste liquid cavity in parallel. The application can realize high-throughput drug screening. Through chip structure design, the chip can be used to generate a detection system containing different drug concentrations. The application can determine whether the to-be-tested drug has an anti-cancer effect and can also determine the influence of the concentration of the drug with an anti-cancer effect on the drug efficacy.

Description

Technical Field

[0001] This invention relates to the field of drug screening, and in particular to a high-throughput microfluidic screening chip and screening method for anticancer drugs. Background Technology

[0002] Malignant tumors are a serious disease that severely damages human health. Anti-cancer drugs are currently the main method for treating cancer. Studying the efficacy and mechanism of action of anti-cancer drugs through in vitro drug screening is of great significance for the research and development of anti-cancer drugs.

[0003] The main challenges in in vitro drug screening currently include the development of highly accurate detection methods or reagents for determining drug efficacy, simplification of detection methods, and improvement of detection throughput.

[0004] Microfluidic chips, with their flexible structural design and low consumption, can improve detection throughput and have been widely used in in vitro drug screening. For example, patent CN104689860B discloses a microfluidic chip and its application for antitumor drug screening at the single-sphere level; CN109894163B discloses a high-throughput, high-content drug screening microfluidic chip and its preparation method; CN101982775B discloses a drug screening method based on cell fluorescence images; and CN110530829A discloses an early drug screening method based on fluorescence.

[0005] Cancer development is caused by enhanced anaerobic glycolysis of glucose within cells coupled with reduced oxygen consumption, a phenomenon known as the Warburg effect (proposed by German physiologist Otto Warburg). Cancer cells typically obtain 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 of glucose. 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 concentration of NAD+ can be measured to reflect the activity of lactate dehydrogenase. Furthermore, by detecting lactate dehydrogenase activity, 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. Designing and synthesizing a detection reagent that can highly sensitively detect the concentration of NAD+ to determine the efficacy of anticancer drugs would be beneficial to promoting the research and development of anticancer drugs, but there are currently few publicly available related plans.

[0006] Therefore, it is now necessary to improve existing technologies to provide more reliable solutions. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a high-throughput anticancer drug microfluidic screening chip and screening method, which addresses the shortcomings of the prior art.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a high-throughput anticancer drug microfluidic screening chip, the screening chip including a chip disk and a plurality of screening channel units arranged in a ring array around the center of the chip disk, which can independently screen anticancer drugs;

[0009] The screening channel unit includes a water inlet, a distribution channel connected to the water inlet, a waste liquid chamber, and several detection channel units connected in parallel between the distribution channel and the waste liquid chamber;

[0010] The detection channel unit includes a quantitative chamber, a first connecting channel, a detection chamber, and a second connecting channel that are sequentially connected to the distribution channel. The end of the second connecting channel is connected to the waste liquid chamber, and the resistance provided by the second connecting channel is greater than the resistance provided by the first connecting channel.

[0011] The detection chamber is pre-packaged with a detection reagent for detecting NAD+ concentration, and the detection chamber is connected to the sample dispensing port through a sample dispensing channel;

[0012] In a single screening channel unit, the volume of each quantitative cavity decreases sequentially from the first direction to the second direction.

[0013] Preferably, the first connecting channel is a long and narrow channel, and the second connecting channel is provided with a serpentine channel section or a centrifugal micro-valve.

[0014] Preferably, both the detection chamber and the waste liquid chamber are connected to an exhaust channel, the end of the exhaust channel is provided with an exhaust port, and a filter membrane is provided at the connection position of the detection chamber and the second connecting channel.

[0015] Preferably, a single screening chip has at least four screening channel units, and each screening channel unit includes at least four detection channel units.

[0016] Preferably, the detection reagent is prepared by the following steps:

[0017] 1) Take 72.5-250.4 mg of dihydropyridine and add it to 50-200 mL of ethanol at a temperature of 40-65℃. Stir for 3-15 min to obtain mixture A;

[0018] 2) Take 91-384 mg of citric acid, 41.7-167 mg of pyridine dicarboxylic acid, and 15-60 mg of cysteine ​​and add them to 100-400 mL of deionized water. Disperse by ultrasonication for 5-30 min to obtain mixture B;

[0019] 3) Add mixture A to mixture B under stirring, and ultrasonically disperse for 15-30 min. Transfer the resulting mixture to a polytetrafluoroethylene-lined reactor and react at 175-200℃ for 6-14 h. After the reaction is completed, cool the mixture and centrifuge the product at 1000-4000 rpm for 2-10 min. Filter the centrifuged liquid through a filter membrane. Dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 700-1400 Da for 24-48 h. Collect the dialysate in the dialysis bag and freeze-dry it to obtain carbon dots.

[0020] 4) Add carbon dots to deionized water and ultrasonically disperse for 5-30 minutes to prepare a carbon dot dispersion with a concentration of 0.25-2 mg / mL;

[0021] 5) Under continuous stirring, add 0.5-2 mL of CuCl2 aqueous solution with a concentration of 1-2.5 mg / mL to 5-20 mL of carbon dot dispersion and mix. Stir for 5-30 min, let stand for 0.5-2 h, centrifuge at 300-800 rpm for 5-20 min, wash the solid product with deionized water, and vacuum dry to obtain the detection reagent.

[0022] Preferably, the detection reagent is prepared by the following steps:

[0023] 1) Add 125.2 mg of dihydropyridine to 100 mL of ethanol at 55 °C and stir for 5 min to obtain mixture A;

[0024] 2) Take 192 mg of citric acid, 83.5 mg of pyridine dicarboxylic acid, and 30 mg of cysteine ​​and add them to 200 mL of deionized water. Disperse by sonication for 10 min to obtain mixture B;

[0025] 3) Add mixture A to mixture B under stirring, and ultrasonically disperse for 30 min. Transfer the resulting mixture to a polytetrafluoroethylene-lined reactor and react at 185 °C for 10 h. After the reaction is completed, cool the mixture and centrifuge the product at 2500 rpm for 3 min. Filter the centrifuged liquid through a 0.20 μm filter membrane. Dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 1000 Da for 36 h. Collect the dialysate in the dialysis bag and freeze-dry it to obtain carbon dots.

[0026] 4) Add carbon dots to deionized water and sonicate for 15 min to prepare a carbon dot dispersion with a concentration of 0.5 mg / mL;

[0027] 5) Under continuous stirring, add 1 mL of CuCl2 aqueous solution with a concentration of 2 mg / mL to 10 mL of carbon dot dispersion, stir for 15 min, let stand for 1 h, centrifuge at 500 rpm for 10 min, wash the solid product with deionized water, and dry under vacuum at 70 °C to obtain the detection reagent.

[0028] This invention also provides a drug screening method, which uses the screening chip described above to screen anticancer drugs in vitro. The method includes the following steps:

[0029] S1. Add deionized water to the water inlet until each quantitative chamber is filled with deionized water, then stop adding water and drive the screening chip to centrifuge at speed R1 for 5-20 seconds, so that the deionized water in the quantitative chamber enters the detection chamber; at speed R1, the deionized water can break through the first flow channel, but cannot break through the second connecting flow channel.

[0030] S2. Then, the screening chip is rotated clockwise and counterclockwise at a speed not greater than R1. The oscillation causes the pre-packaged detection reagent in the detection chamber to disperse in deionized water.

[0031] The reciprocating rotation program is as follows: clockwise rotation at 100-200 rpm for 3-15 seconds, counterclockwise rotation at 100-200 rpm for 3-15 seconds; repeat 5-20 times.

[0032] S3. Add a quantitative amount of cancer cells and the drug to be tested to the sample loading port, then add a quantitative amount of deionized water to rinse the sample loading port and the sample loading channel, drive the screening chip to centrifuge at a speed of R0 for 5-30 seconds, and then rotate it clockwise and counterclockwise alternately at a speed not greater than R0 to make the detection system in the detection chamber mix evenly.

[0033] The reciprocating rotation program is as follows: clockwise rotation at 80-150 rpm for 3-15 seconds, counterclockwise rotation at 80-150 rpm for 3-15 seconds; repeat 5-20 times.

[0034] S4. Place the screening chip at 37℃ and incubate for 1-4 hours;

[0035] S5. Centrifuge at R2 speed for 15-60s to allow the liquid in the detection chamber to break through the second flow channel and enter the waste liquid chamber.

[0036] Then add deionized water to the sample loading port, centrifuge at R2 speed for 10-60s to rinse the detection chamber, and repeat rinsing 2-5 times;

[0037] Where R0 < R1 < R2;

[0038] S6. Irradiate the detection cavity of the screening chip under 328nm excitation light, and detect the fluorescence intensity at 472nm and 633nm, respectively, and record them as F. 472 F 633 Calculate the ratio K, K = F 472 / F 633Then, based on the pre-established standard curve f representing the relationship between the value of K and the NAD+ concentration, the NAD+ concentration is analyzed and obtained. Finally, the anticancer efficacy of the drug to be tested is determined based on the NAD+ concentration.

[0039] The smaller the value of K, the lower the NAD+ concentration, indicating that the drug being tested has a better anticancer effect.

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

[0041] S1. Add deionized water to the water inlet until each quantitative chamber is filled with deionized water, then stop adding water and drive the screening chip to centrifuge at a speed of R1 for 10 seconds, so that the deionized water in the quantitative chamber enters the detection chamber; at a speed of R1, the deionized water can break through the first flow channel, but cannot break through the second connecting flow channel.

[0042] S2. Then, the screening chip is rotated clockwise and counterclockwise at a speed not greater than R1. The oscillation causes the pre-packaged detection reagent in the detection chamber to be dispersed in deionized water.

[0043] The reciprocating rotation program is as follows: clockwise rotation at 150 rpm for 5 seconds, counterclockwise rotation at 150 rpm for 5 seconds; repeat 10 times.

[0044] S3. Add a quantitative amount of cancer cells and the drug to be tested to the sample inlet, then add a quantitative amount of deionized water to rinse the sample inlet and sample flow channel, drive the screening chip to centrifuge at a speed of R0 for 10s, and then rotate clockwise and counterclockwise alternately at a speed not greater than R0 to make the detection system in the detection chamber mix evenly.

[0045] The reciprocating rotation program is as follows: clockwise rotation for 5 seconds at 100 rpm, counterclockwise rotation for 5 seconds at 100 rpm; repeat 10 times.

[0046] S4. Incubate the screening chip at 37°C for 1.5 hours;

[0047] S5. Centrifuge at R2 speed for 30s to allow the liquid in the detection chamber to break through the second flow channel and enter the waste liquid chamber.

[0048] Then add deionized water to the sample loading port, centrifuge at R2 speed for 30s to rinse the detection chamber, and repeat the rinsing 3 times;

[0049] Where R0 < R1 < R2;

[0050] S6. Irradiate the detection cavity of the screening chip under 328nm excitation light, and detect the fluorescence intensity at 472nm and 633nm, respectively, and record them as F. 472 F 633 Calculate the ratio K, K = F472 / F 633 Then, based on the pre-established standard curve f representing the relationship between the value of K and the NAD+ concentration, the NAD+ concentration is analyzed and obtained. Finally, the anticancer efficacy of the drug to be tested is determined based on the NAD+ concentration.

[0051] The smaller the value of K, the lower the NAD+ concentration, indicating that the drug being tested has a better anticancer effect.

[0052] Preferably, the standard curve f is constructed by the following method:

[0053] 1) Add the test reagent to deionized water and ultrasonically disperse for 5-30 minutes to prepare a test reagent dispersion with a concentration of 0.1-2 mg / mL;

[0054] 2) Add different amounts of NAD+ to the detection reagent dispersion, stir evenly, and prepare a series of standard working solutions with the same detection reagent concentration but different NAD+ concentrations. The NAD+ concentration range in this series of standard working solutions is 0-5 μmol / L.

[0055] 3) Place the standard working solution at 37℃ and let it stand for 1-4 hours. Centrifuge and discard the centrifuged liquid. Add the obtained solid to deionized water to prepare a dispersion. Irradiate the dispersion under an excitation light of 328 nm and detect the fluorescence intensity at 472 nm and 633 nm, respectively, and record them as F. 472 F 633 Calculate the ratio K, K = F 472 / F 633 , with K B Using the x-axis as the horizontal axis and the NAD+ concentration as the y-axis, curve fitting is performed to obtain the standard curve f.

[0056] Preferably, R0 = 80-350 rpm, R1 = 500-1750 rpm, and R2 = 1800-3000 rpm.

[0057] The beneficial effects of this invention are:

[0058] This invention provides a high-throughput microfluidic screening chip for anticancer drugs and a method for screening anticancer drugs based on the chip. This invention can achieve high-throughput drug screening. Through chip structure design, the chip can be used to generate a detection system containing different drug concentrations. It can determine whether the drug to be tested has anticancer effects, and at the same time, it can also determine the effect of the concentration of the anticancer drug on the efficacy.

[0059] In this invention, by pre-encapsulating the detection reagents within a chip and automatically generating detection systems for different drug concentrations using the chip, the detection procedure can be simplified and the detection efficiency improved. Furthermore, the screening method of this invention is highly accurate, simple to operate, and has excellent prospects for widespread application.

[0060] The detection reagent of this invention determines the inhibitory effect of the drug on lactate dehydrogenase activity by detecting the concentration of NAD+, a marker of lactate dehydrogenase activity, thereby enabling the screening of the anticancer efficacy of the drug. This detection reagent is based on fluorescence analysis, using the ratio of blue fluorescence to red fluorescence to analyze the NAD+ concentration and thus determine the efficacy, which can reduce the influence of background fluorescence and improve the anti-interference ability and sensitivity of the detection results. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of the high-throughput anticancer drug microfluidic screening chip of the present invention;

[0062] Figure 2 This is a schematic diagram of the screening channel unit of the present invention;

[0063] Figure 3 This is a schematic diagram showing the placement of the filter membrane within the waste liquid chamber of the present invention.

[0064] Figure 4 The infrared spectrum of the carbon dots prepared in Example 1 of this invention;

[0065] Figure 5 The image shows the ultraviolet-visible absorption spectrum of the carbon dots prepared in Example 1 of this invention.

[0066] Figure 6 The emission spectrum of the carbon dots prepared in Example 1 of the present invention at 328 nm is shown.

[0067] Figure 7 Cu in this invention 2+ The quenching results of the blue fluorescence of carbon dots;

[0068] Figure 8 The fluorescence response of the detection reagent prepared in Example 1 of the present invention to different concentrations of NAD+;

[0069] Figure 9 This is the standard curve constructed in this invention;

[0070] Figure 10 The results of the cell toxicity test of the detection reagent prepared in Example 1 of the present invention.

[0071] Explanation of reference numerals in the attached figures:

[0072] 1—Chip disk; 2—Screening channel unit; 3—Drive hole; 4—Water inlet; 5—Dispensing channel; 6—Detection channel unit; 7—Waste liquid chamber; 8—Detection reagent; 61—Quantitative chamber; 62—First connecting channel; 63—Detection chamber; 64—Second connecting channel; 65—Sample dispensing channel; 66—Sample dispensing port; 67—Exhaust channel; 68—Exhaust port; 631—Filter membrane; 641—Serpentine channel section. Detailed Implementation

[0073] 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.

[0074] 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.

[0075] The present invention provides a high-throughput anticancer drug microfluidic screening chip, which includes a chip disk 1 and a plurality of screening channel units 2 arranged in a ring array around the center of the chip disk 1, which can independently screen anticancer drugs.

[0076] The screening channel unit 2 includes a water inlet 4, a distribution channel 5 connected to the water inlet 4, a waste liquid chamber, and a plurality of detection channel units 6 connected in parallel between the distribution channel 5 and the waste liquid chamber;

[0077] The detection channel unit 6 includes a quantitative cavity 61, a first connecting channel 62, a detection cavity 63, and a second connecting channel 64 that are sequentially connected to the distribution channel 5. The end of the second connecting channel 64 is connected to the waste liquid cavity, and the resistance provided by the second connecting channel 64 is greater than the resistance provided by the first connecting channel 62.

[0078] The detection chamber 63 is pre-encapsulated with a detection reagent 8 for detecting NAD+ concentration, and the detection chamber 63 is connected to the sample dispensing port 66 through the sample dispensing channel 65.

[0079] In a single screening channel unit 2, from the first direction to the second direction (e.g.) Figure 2 (From left to right in the middle), the volume of each quantitative cavity 61 decreases sequentially.

[0080] In a preferred embodiment, a drive hole 3 is provided in the middle of the chip disk 1 to facilitate drive connection to the centrifugal drive structure for centrifugation.

[0081] In a preferred embodiment, the first connecting channel 62 is a long and narrow channel, and the second connecting channel 64 is provided with a serpentine channel section 641 or a centrifugal microvalve to provide a certain resistance through the long and narrow channel, the serpentine channel section 641 or the centrifugal microvalve, so that when a certain centrifugal force (i.e., centrifugal speed) is met, the liquid can break through the corresponding flow and enter the downstream.

[0082] More specifically, in this embodiment, the resistance provided by the first connecting channel 62 is less than that of the second connecting channel 64, meaning that the centrifugal speed required for the liquid to break through the second connecting channel 64 is greater than that of the first connecting channel 62. The resistance provided can be adjusted by setting the length and width of the connecting flow, and by selecting different centrifugal microvalves, so that the first connecting channel 62 and the second connecting channel 64 can meet the above conditions.

[0083] In a preferred embodiment, both the detection chamber 63 and the waste liquid chamber are connected to an exhaust channel 67, and an exhaust port 68 is provided at the end of the exhaust channel 67. A filter membrane 631 is provided at the connection position of the detection chamber 63 and the second connecting channel 64.

[0084] In a preferred embodiment, a single screening chip is provided with at least four screening channel units 2, and each screening channel unit 2 includes at least four detection channel units 6.

[0085] In a preferred embodiment, the detection reagent is prepared by the following steps:

[0086] 1) Take 72.5-250.4 mg of dihydropyridine and add it to 50-200 mL of ethanol at a temperature of 40-65℃. Stir for 3-15 min to obtain mixture A;

[0087] 2) Take 91-384 mg of citric acid, 41.7-167 mg of pyridine dicarboxylic acid, and 15-60 mg of cysteine ​​and add them to 100-400 mL of deionized water. Disperse by ultrasonication for 5-30 min to obtain mixture B;

[0088] 3) Add mixture A to mixture B under stirring, and ultrasonically disperse for 15-30 min. Transfer the resulting mixture to a polytetrafluoroethylene-lined reactor and react at 175-200℃ for 6-14 h. After the reaction is completed, cool the mixture and centrifuge the product at 1000-4000 rpm for 2-10 min. Filter the centrifuged liquid through a 631 filter membrane. Dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 700-1400 Da for 24-48 h. Collect the dialysate in the dialysis bag and freeze-dry it to obtain carbon dots.

[0089] 4) Add carbon dots to deionized water and ultrasonically disperse for 5-30 minutes to prepare a carbon dot dispersion with a concentration of 0.25-2 mg / mL;

[0090] 5) Under continuous stirring, add 0.5-2 mL of CuCl2 aqueous solution with a concentration of 1-2.5 mg / mL to 5-20 mL of carbon dot dispersion and mix. Stir for 5-30 min, let stand for 0.5-2 h, centrifuge at 300-800 rpm for 5-20 min, wash the solid product with deionized water, and vacuum dry to obtain the detection reagent.

[0091] The detection reagent of the present invention determines the inhibitory effect of the drug on lactate dehydrogenase activity by detecting the concentration of NAD+, a marker of lactate dehydrogenase activity, thereby enabling the screening of the anticancer efficacy of the drug.

[0092] Cancer development is caused by enhanced anaerobic glycolysis within cells coupled with reduced oxygen consumption, a phenomenon known as the Warburg effect. 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 of glucose. In cancer cells where the metabolic rate is too rapid, leading to lactate accumulation, the concentration of NAD+ in cancer cells is far higher than in normal cells.

[0093] Therefore, the activity of lactate dehydrogenase can be reflected by measuring the concentration of NAD+. When screening anticancer drugs, the inhibitory effect of anticancer drugs on lactate dehydrogenase activity can be determined by detecting the activity of lactate dehydrogenase. Thus, the detection of NAD+ concentration can be used to screen the efficacy of anticancer drugs.

[0094] The main principles of the present invention will be explained below to facilitate understanding of the invention.

[0095] This invention first synthesizes a carbon dot CDs using citric acid, pyridine dicarboxylic acid, cysteine, and 2,6-dimethyl-3,5-diethyl-1,4-dihydropyridine as main raw materials via a hydrothermal method. The carbon dots are then mixed with CuCl2 solution, centrifuged, and dried to obtain the detection reagent. This carbon dot exhibits dual fluorescence emission characteristics, emitting bright blue fluorescence (emission peak near 472 nm) and red fluorescence (emission peak near 633 nm) under excitation light at approximately 328 nm. The red fluorescence originates from the sp atoms within the carbon core. 2 The inherent band gap generated by the conjugated structure and the blue fluorescence originate from the defect state luminescence caused by the incomplete carbonization of 2,6-dimethyl-3,5-diethyl ester-1,4-dihydropyridine residues on the carbon dot surface.

[0096] Furthermore, the surface of these carbon dots is rich in functional groups such as carboxyl, hydroxyl, thiol, and amino groups, and they exhibit good water solubility and biocompatibility. The carboxyl, hydroxyl, thiol, and amino functional groups on the surface of the carbon dots interact with Cu. 2+ It has good complexation properties, and Cu 2+ After complexing with carbon dots, the blue fluorescence caused by defect-state luminescence is quenched due to excited-state electron and energy transfer, while the red fluorescence originating from the carbon nucleus remains unaffected; when NAD+ is present in the system, the phosphate groups on NAD+ affect Cu. 2+ It has stronger coordination ability, thus competitively acquiring Cu from carbon dots. 2+ Cu@NAD+ coordination compounds are formed. Centrifugation separates these coordination compounds from the carbon dots, and the blue fluorescence of the carbon dots is restored. The intensity of the restored blue fluorescence is positively correlated with the NAD+ concentration. Calculating the NAD+ concentration by using the ratio of blue fluorescence to red fluorescence reduces the influence of background fluorescence, improving the anti-interference ability and sensitivity of the detection results.

[0097] The carbon dots are doped with elements such as sulfur and nitrogen, which increases the electron cloud density and thus enhances fluorescence intensity. Furthermore, the temporary occupancy of sulfur during carbon dot synthesis increases the proportion of pyrrole N in the carbon dots. Pyrrole N imparts appropriate selective adsorption properties for NAD+ to the carbon dots, allowing NAD+ to be adsorbed onto the carbon dot surface. This facilitates the efficient competitive acquisition of Cu complexes by NAD+ from the carbon dots. 2+ The centrifugation process produces a blue fluorescence response.

[0098] By detecting the fluorescence intensity ratio K and calculating the NAD+ concentration using a standard curve, the efficacy of the test drug in inhibiting lactate dehydrogenase activity can be determined. The lower the NAD+ concentration, the better the efficacy in inhibiting lactate dehydrogenase activity, which means the better the anti-cancer effect of the test drug.

[0099] This invention also provides a drug screening method, which uses the screening chip described above to screen anticancer drugs in vitro. The method includes the following steps:

[0100] S1. Add deionized water to the water inlet 4 until each quantitative chamber 61 is filled with deionized water, then stop adding water and drive the screening chip to centrifuge at a speed of R1 for 5-20 seconds, so that the deionized water in the quantitative chamber 61 enters the detection chamber 63. At a speed of R1, the deionized water can break through the first flow channel and enter the detection chamber 63, but cannot break through the second connecting flow channel 64.

[0101] S2. Then, the screening chip is rotated clockwise and counterclockwise at a speed not greater than R1, and the detection reagent pre-packaged in the detection chamber 63 is dispersed in deionized water through the oscillation.

[0102] The reciprocating rotation program is as follows: clockwise rotation at 100-200 rpm for 3-15 seconds, counterclockwise rotation at 100-200 rpm for 3-15 seconds; repeat 5-20 times.

[0103] S3. Add a quantitative amount of cancer cells and the drug to be tested to the sample inlet 66, and then add a quantitative amount of deionized water to rinse the sample inlet 66 and the sample flow channel 65, so that the cancer cells and the drug to be tested enter the detection chamber 63. Drive the screening chip to centrifuge at a speed of R0 for 5-30 seconds, and then rotate it clockwise and counterclockwise alternately at a speed not greater than R0 to make the detection system in the detection chamber 63 mix evenly.

[0104] The reciprocating rotation program is as follows: clockwise rotation at 80-150 rpm for 3-15 seconds, counterclockwise rotation at 80-150 rpm for 3-15 seconds; repeat 5-20 times.

[0105] S4. Place the screening chip at 37℃ and incubate for 1-4 hours;

[0106] S5. Centrifuge at R2 speed for 15-60s to allow the liquid in the detection chamber 63 to break through the second flow channel and enter the waste liquid chamber. Due to the effect of the filter membrane 631 (the filter pore size of the filter membrane 631 is smaller than the particle size of the detection reagent and cancer cells), the detection reagent and cancer cells will be retained in the detection chamber 63, and only the liquid (deionized water, excess drugs, etc.) will be discharged into the waste liquid chamber.

[0107] Then add deionized water to sample port 66, centrifuge at R2 speed for 10-60s, rinse detection chamber 63, repeat rinsing 2-5 times;

[0108] Where R0 < R1 < R2;

[0109] S6. Irradiate the detection cavity 63 of the screening chip under 328nm excitation light, and detect the fluorescence intensity at 472nm and 633nm, respectively, and record them as F. 472 F 633 Calculate the ratio K, K = F 472 / F 633 Then, based on the pre-established standard curve f representing the relationship between the value of K and the NAD+ concentration, the NAD+ concentration is analyzed and obtained. Finally, the anticancer efficacy of the drug to be tested is determined based on the NAD+ concentration.

[0110] The smaller the value of K, the lower the NAD+ concentration, indicating that the drug being tested has a better anticancer effect.

[0111] In a preferred embodiment, the standard curve f is constructed by the following method:

[0112] 1) Add the test reagent to deionized water and ultrasonically disperse for 5-30 minutes to prepare a test reagent dispersion with a concentration of 0.1-2 mg / mL;

[0113] 2) Add different amounts of NAD+ to the detection reagent dispersion, stir evenly, and prepare a series of standard working solutions with the same detection reagent concentration but different NAD+ concentrations. The NAD+ concentration range in this series of standard working solutions is 0-5 μmol / L.

[0114] 3) Place the standard working solution at 37℃ and let it stand for 1-4 hours. Centrifuge and discard the centrifuged liquid. Add the obtained solid to deionized water to prepare a dispersion. Irradiate the dispersion under an excitation light of 328 nm and detect the fluorescence intensity at 472 nm and 633 nm, respectively, and record them as F. 472 F 633 Calculate the ratio K, K = F 472 / F 633 , with K B Using the x-axis as the horizontal axis and the NAD+ concentration as the y-axis, curve fitting is performed to obtain the standard curve f.

[0115] In a preferred embodiment, R0 = 80-350 rpm, R1 = 500-1750 rpm, and R2 = 1800-3000 rpm.

[0116] The cancer cells used can be any one of the following: HeLa cells, human lung cancer cells Calu-1, human lung cancer cells A549, human lung squamous cell carcinoma cells SK-MES-1, human colon cancer cells HT-29, human colon cancer cells SW480, human liver cancer cells Hep G2, human breast cancer cells MDA-MB-231, human prostate cancer cells PC-3, or human prostate cancer cells 22RV1.

[0117] The main principles of chip screening are explained below:

[0118] Reference Figure 2Taking a single screening channel unit 2 as an example, the volumes of the four detection chambers 63 decrease sequentially from left to right. Therefore, in step S2, the volume of deionized water entering the four detection chambers 63 from left to right at rotation speed R1 will decrease sequentially. (During this process, a small amount of deionized water accumulated in the distribution channel 5 will also enter the detection chambers 63 under centrifugal force, but since its amount is small and it is basically evenly distributed into the four detection chambers 63, it will not affect the distribution pattern of the deionized water in the four detection chambers 63 decreasing sequentially.) In step S3, the amount of cancer cells, test drugs, and deionized water added to the four detection chambers 63 is the same. Therefore, the final result is that the concentration of test drugs in the four detection chambers 63 from left to right will increase sequentially. That is, several detection systems with gradient drug concentrations are obtained in one screening channel unit 2. After cultivation and NAD+ concentration detection, the anticancer efficacy of the test drug can be detected, and the inhibitory effect of different concentrations of test drugs on cancer cells can also be detected. Furthermore, a single screening chip is equipped with multiple screening channels, enabling the simultaneous screening of multiple drugs to be tested. Therefore, the screening chip of this invention has high throughput characteristics. At the same time, it can also generate detection systems with different drug concentrations through a special chip structure. It can determine whether a drug to be tested has anti-cancer effects, and at the same time obtain the effect of the concentration of anti-cancer drugs on the efficacy.

[0119] Example 1: Preparation of Detection Reagent

[0120] 1) Add 125.2 mg of dihydropyridine to 100 mL of ethanol at 55 °C and stir for 5 min to obtain mixture A;

[0121] 2) Take 192 mg of citric acid, 83.5 mg of pyridine dicarboxylic acid, and 30 mg of cysteine ​​and add them to 200 mL of deionized water. Disperse by sonication for 10 min to obtain mixture B;

[0122] 3) Add mixture A to mixture B under stirring, and ultrasonically disperse for 30 min. Transfer the resulting mixture to a polytetrafluoroethylene-lined reactor and react at 185 °C for 10 h. After the reaction is completed, cool the mixture and centrifuge the product at 2500 rpm for 3 min. Filter the centrifuged liquid through a 0.20 μm 631 filter membrane. Dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 1000 Da for 36 h. Collect the dialysate in the dialysis bag and freeze-dry it to obtain carbon dots.

[0123] 4) Add carbon dots to deionized water and sonicate for 15 min to prepare a carbon dot dispersion with a concentration of 0.5 mg / mL;

[0124] 5) Under continuous stirring, add 1 mL of CuCl2 aqueous solution with a concentration of 2 mg / mL to 10 mL of carbon dot dispersion, stir for 15 min, let stand for 1 h, centrifuge at 500 rpm for 10 min, wash the solid product with deionized water, and dry under vacuum at 70 °C to obtain the detection reagent.

[0125] Performance testing and characterization

[0126] Reference Figure 4 The image shows the infrared spectrum of the carbon dots (CDs) prepared in Example 1, with the inset being a transmission electron microscope (TEM) image of the CDs. Analysis of the infrared spectrum shows that the surface is rich in carboxyl, amino, carbonyl, and thiol groups. According to the TEM image, the average size of the carbon dots is about 10 nm.

[0127] Reference Figure 5 The image shows the UV-Vis absorption spectrum of the carbon dots prepared in Example 1. It can be seen that they have a wide absorption range, with the maximum absorption peak located near 328 nm.

[0128] Reference Figure 6 The image shows the emission spectrum of the carbon dots prepared in Example 1 at 328 nm excitation light. It can be seen that they have dual fluorescence properties, with emission peaks near 472 nm and 633 nm.

[0129] Reference Figure 7 , is Cu 2+ The quenching results of the blue fluorescence of carbon dots were obtained by preparing a 1 mg / mL carbon dot dispersion and adding equal volumes of CuCl2 aqueous solutions with a certain concentration gradient (0, 1, 2, 3, 4, 6, 8, 10 mg / mL). The fluorescence intensity at 472 nm and 633 nm was then measured under excitation light of 328 nm. The results show that the blue fluorescence intensity gradually decreases with increasing CuCl2 concentration, while the red fluorescence remains unaffected, indicating that CuCl2 quenching... 2+ Blue fluorescence capable of quenching carbon dots.

[0130] Reference Figure 8 The fluorescence response of the detection reagent prepared in Example 1 to different concentrations of NAD+ is shown below. First, the prepared detection reagent was added to deionized water and ultrasonically dispersed. Then, different amounts of NAD+ were added and stirred until homogeneous, resulting in a series of standard working solutions containing the same detection reagent concentration (1 mg / mL) but different NAD+ concentrations (0, 1, 2, 3, 4, 5 μmol / L). The standard working solutions were allowed to stand at 37°C for 2 hours, then centrifuged. The centrifuged liquid was discarded, and the resulting solid was added to deionized water to prepare dispersions of the same concentration. The dispersions were then placed under excitation light at 328 nm, and the fluorescence intensity at 472 nm and 633 nm was measured. The test results are shown below. Figure 8As shown, it can be seen that with the increase of NAD+ concentration, the intensity of blue fluorescence at 472nm gradually increases, while the intensity of red fluorescence at 633nm remains unaffected, indicating that NAD+ can restore the blue fluorescence of the detection reagent.

[0131] The fluorescence intensities at 472 nm and 633 nm are denoted as F, respectively. B472 F B633 Calculate the ratio K B K B =F B472 / F B633 , with K B Using the x-axis as the horizontal axis and NAD+ concentration as the y-axis, a curve was fitted to obtain a standard curve, as shown in the figure. Figure 9 As shown, the relevance R 2 =0.9992, indicating a good linear relationship.

[0132] Reference Figure 10 The results of the cytotoxicity test on the reagent prepared in Example 1 are shown below. HeLa cells were used as the experimental subject, and the MTT Cell Proliferation and Cytotoxicity Assay Kit was employed to detect the toxicity of the reagent. The horizontal axis represents the concentration of the reagent, and the vertical axis represents cell viability. The test results show that the reagent has very low cytotoxicity.

[0133] Example 2

[0134] Reference Figure 1-3 This embodiment provides a high-throughput anticancer drug microfluidic screening chip, which includes a chip disk 1 and four screening channel units 2 arranged in a ring array around the center of the chip disk 1, which can independently screen anticancer drugs.

[0135] The screening channel unit 2 includes a water inlet 4, a distribution channel 5 connected to the water inlet 4, a waste liquid chamber, and four detection channel units 6 connected in parallel between the distribution channel 5 and the waste liquid chamber.

[0136] The detection channel unit 6 includes a quantitative cavity 61, a first connecting channel 62, a detection cavity 63, and a second connecting channel 64 that are sequentially connected to the distribution channel 5. The end of the second connecting channel 64 is connected to the waste liquid cavity, and the resistance provided by the second connecting channel 64 is greater than the resistance provided by the first connecting channel 62.

[0137] The detection chamber 63 is pre-encapsulated with a detection reagent 8 for detecting NAD+ concentration, and the detection chamber 63 is connected to the sample dispensing port 66 through the sample dispensing channel 65.

[0138] In a single screening channel unit 2, from the first direction to the second direction (e.g.) Figure 2 (From left to right in the middle), the volume of each quantitative cavity 61 decreases sequentially.

[0139] The chip disk 1 has a drive hole 3 in the middle so that it can be connected to the centrifugal drive structure for centrifugal operation.

[0140] The first connecting channel 62 is an elongated channel, and the second connecting channel 64 has a serpentine flow section 641. The elongated channel and the serpentine flow section 641 provide resistance, allowing the liquid to overcome the required centrifugal force (i.e., centrifugal speed) and flow downstream. The resistance provided by the first connecting channel 62 is less than that of the second connecting channel 64, meaning the centrifugal speed required for the liquid to overcome the second connecting channel 64 is greater than that required for the first connecting channel 62.

[0141] The detection chamber 63 and the waste liquid chamber are both connected to an exhaust channel 67. An exhaust port 68 is provided at the end of the exhaust channel 67. A filter membrane 631 is provided at the connection position of the detection chamber 63 and the second connecting channel 64. The filter membrane 631 is a nano filter membrane 631 with a pore size of 4nm.

[0142] Example 3

[0143] A drug screening method, which uses the screening chip of Example 2 to screen anticancer drugs, specifically includes the following steps:

[0144] S1. Add deionized water to the water inlet 4 until each quantitative chamber 61 is filled with deionized water, then stop adding water and drive the screening chip to centrifuge at a speed of R1 for 10 seconds, so that the deionized water in the quantitative chamber 61 enters the detection chamber 63; at a speed of R1, the deionized water can break through the first flow channel, but cannot break through the second connecting flow channel 64.

[0145] S2. Then, the screening chip is rotated clockwise and counterclockwise at a speed not greater than R1. The oscillation causes the detection reagent pre-packaged in the detection chamber 63 to be dispersed in deionized water.

[0146] The reciprocating rotation program is as follows: clockwise rotation at 150 rpm for 5 seconds, counterclockwise rotation at 150 rpm for 5 seconds; repeat 10 times.

[0147] S3. Add a quantitative amount of cancer cells and the drug to be tested to the sample loading port 66, then add a quantitative amount of deionized water to rinse the sample loading port 66 and the sample loading channel 65, drive the screening chip to centrifuge at a speed of R0 for 10s, and then rotate clockwise and counterclockwise alternately at a speed not greater than R0 to make the detection system in the detection chamber 63 mixed evenly.

[0148] The reciprocating rotation program is as follows: clockwise rotation for 5 seconds at 100 rpm, counterclockwise rotation for 5 seconds at 100 rpm; repeat 10 times.

[0149] S4. Incubate the screening chip at 37°C for 1.5 hours;

[0150] S5. Centrifuge at R2 speed for 30s to allow the liquid in detection chamber 63 to break through the second flow channel and enter the waste liquid chamber;

[0151] Then add deionized water to sample port 66, centrifuge at R2 speed for 30s to rinse detection chamber 63, and repeat rinsing 3 times;

[0152] Where R0 = 200 rpm, R1 = 1000 rpm, and R2 = 2100 rpm;

[0153] S6. Irradiate the detection cavity 63 of the screening chip under 328nm excitation light, and detect the fluorescence intensity at 472nm and 633nm, respectively, and record them as F. 472 F 633 Calculate the ratio K, K = F 472 / F 633 Then, based on the pre-established standard curve f representing the relationship between the value of K and the NAD+ concentration, the NAD+ concentration is analyzed and obtained. Finally, the anticancer efficacy of the drug to be tested is determined based on the NAD+ concentration.

[0154] The smaller the value of K, the lower the NAD+ concentration, indicating that the drug being tested has a better anticancer effect.

[0155] 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 high-throughput microfluidic screening chip for anticancer drugs, characterized in that, The screening chip includes a chip disk and several screening channel units arranged in a ring array around the center of the chip disk, which can independently screen anticancer drugs. The screening channel unit includes a water inlet, a distribution channel connected to the water inlet, a waste liquid chamber, and several detection channel units connected in parallel between the distribution channel and the waste liquid chamber; The detection channel unit includes a quantitative chamber, a first connecting channel, a detection chamber, and a second connecting channel that are sequentially connected to the distribution channel. The end of the second connecting channel is connected to the waste liquid chamber, and the resistance provided by the second connecting channel is greater than the resistance provided by the first connecting channel. The detection chamber is pre-packaged with a detection reagent for detecting NAD+ concentration, and the detection chamber is connected to the sample dispensing port through a sample dispensing channel; In a single screening channel unit, the volume of each quantitative cavity decreases sequentially from the first direction to the second direction; The detection reagent is prepared by the following steps: 1) Take 72.5-250.4 mg of dihydropyridine and add it to 50-200 mL of ethanol at a temperature of 40-65℃. Stir for 3-15 min to obtain mixture A; 2) Take 91-384 mg of citric acid, 41.7-167 mg of pyridine dicarboxylic acid, and 15-60 mg of cysteine ​​and add them to 100-400 mL of deionized water. Disperse by ultrasonication for 5-30 min to obtain mixture B; 3) Add mixture A to mixture B under stirring, and ultrasonically disperse for 15-30 min. Transfer the resulting mixture to a polytetrafluoroethylene-lined reactor and react at 175-200℃ for 6-14 h. After the reaction is completed, cool the mixture and centrifuge the product at 1000-4000 rpm for 2-10 min. Filter the centrifuged liquid through a filter membrane. Dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 700-1400 Da for 24-48 h. Collect the dialysate in the dialysis bag and freeze-dry it to obtain carbon dots. 4) Add carbon dots to deionized water and ultrasonically disperse for 5-30 minutes to prepare a carbon dot dispersion with a concentration of 0.25-2 mg / mL; 5) Under continuous stirring, add 0.5-2 mL of CuCl2 aqueous solution with a concentration of 1-2.5 mg / mL to 5-20 mL of carbon dot dispersion and mix. Stir for 5-30 min, let stand for 0.5-2 h, centrifuge at 300-800 rpm for 5-20 min, wash the solid product with deionized water, and vacuum dry to obtain the detection reagent.

2. The high-throughput anticancer drug microfluidic screening chip according to claim 1, characterized in that, The first connecting channel is a long and narrow channel, and the second connecting channel is provided with a serpentine channel section or a centrifugal micro-valve.

3. The high-throughput anticancer drug microfluidic screening chip according to claim 2, characterized in that, Both the detection chamber and the waste liquid chamber are connected to an exhaust channel, and an exhaust port is provided at the end of the exhaust channel. A filter membrane is provided at the connection position of the detection chamber and the second connecting channel.

4. The high-throughput anticancer drug microfluidic screening chip according to claim 3, characterized in that, Each screening chip has at least four screening channel units, and each screening channel unit includes at least four detection channel units.

5. The high-throughput anticancer drug microfluidic screening chip according to claim 1, characterized in that, The detection reagent is prepared by the following steps: 1) Take 125.2 mg of dihydropyridine and add it to 100 mL of ethanol at 55 °C. Stir for 5 min to obtain mixture A; 2) Take 192 mg of citric acid, 83.5 mg of pyridine dicarboxylic acid, and 30 mg of cysteine ​​and add them to 200 mL of deionized water. Disperse by sonication for 10 min to obtain mixture B; 3) Add mixture A to mixture B under stirring, and ultrasonically disperse for 30 min. Transfer the resulting mixture to a polytetrafluoroethylene-lined reactor and react at 185 °C for 10 h. After the reaction is completed, cool the mixture and centrifuge the product at 2500 rpm for 3 min. Filter the centrifuged liquid through a 0.20 μm filter membrane. Dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 1000 Da for 36 h. Collect the dialysate in the dialysis bag and freeze-dry it to obtain carbon dots. 4) Add carbon dots to deionized water and sonicate for 15 min to prepare a carbon dot dispersion with a concentration of 0.5 mg / mL; 5) Under continuous stirring, add 1 mL of CuCl2 aqueous solution with a concentration of 2 mg / mL to 10 mL of carbon dot dispersion, stir for 15 min, let stand for 1 h, centrifuge at 500 rpm for 10 min, wash the solid product with deionized water, and dry under vacuum at 70 °C to obtain the detection reagent.

6. A drug screening method, characterized in that, The method employs a screening chip as described in any one of claims 1-5 for in vitro screening of anticancer drugs, and includes the following steps: S1. Add deionized water to the water inlet until each quantitative chamber is filled with deionized water, then stop adding water and drive the screening chip to centrifuge at speed R1 for 5-20 seconds, so that the deionized water in the quantitative chamber enters the detection chamber; at speed R1, the deionized water can break through the first flow channel, but cannot break through the second connecting flow channel. S2. Then, the screening chip is rotated clockwise and counterclockwise at a speed not greater than R1. The oscillation causes the pre-packaged detection reagent in the detection chamber to disperse in deionized water. The reciprocating rotation program is as follows: clockwise rotation at 100-200 rpm for 3-15 seconds, counterclockwise rotation at 100-200 rpm for 3-15 seconds; repeat 5-20 times. S3. Add a quantitative amount of cancer cells and the drug to be tested to the sample loading port, then add a quantitative amount of deionized water to rinse the sample loading port and the sample loading channel, drive the screening chip to centrifuge at a speed of R0 for 5-30 seconds, and then rotate it clockwise and counterclockwise alternately at a speed not greater than R0 to make the detection system in the detection chamber mix evenly. The reciprocating rotation program is as follows: clockwise rotation at 80-150 rpm for 3-15 seconds, counterclockwise rotation at 80-150 rpm for 3-15 seconds; repeat 5-20 times. S4. Place the screening chip at 37℃ and incubate for 1-4 hours; S5. Centrifuge at R2 speed for 15-60s to allow the liquid in the detection chamber to break through the second flow channel and enter the waste liquid chamber. Then add deionized water to the sample loading port, centrifuge at R2 speed for 10-60s to rinse the detection chamber, and repeat rinsing 2-5 times; Where R0 < R1 < R2; S6. Irradiate the detection cavity of the screening chip under 328nm excitation light, and detect the fluorescence intensity at 472nm and 633nm, respectively, and record them as F. 472 F 633 Calculate the ratio K, K=F 472 / F 633 Then, based on a pre-established standard curve characterizing the relationship between the value of K and NAD+ concentration... f The NAD+ concentration was analyzed, and the anticancer efficacy of the drug was determined based on the NAD+ concentration. The smaller the value of K, the lower the NAD+ concentration, indicating that the drug being tested has a better anticancer effect.

7. The drug screening method according to claim 6, characterized in that, The method includes the following steps: S1. Add deionized water to the water inlet until each quantitative chamber is filled with deionized water, then stop adding water and drive the screening chip to centrifuge at a speed of R1 for 10 seconds, so that the deionized water in the quantitative chamber enters the detection chamber; at a speed of R1, the deionized water can break through the first flow channel, but cannot break through the second connecting flow channel. S2. Then, the screening chip is rotated clockwise and counterclockwise at a speed not greater than R1. The oscillation causes the pre-packaged detection reagent in the detection chamber to be dispersed in deionized water. The reciprocating rotation program is as follows: clockwise rotation at 150 rpm for 5 seconds, counterclockwise rotation at 150 rpm for 5 seconds; repeat 10 times. S3. Add a quantitative amount of cancer cells and the drug to be tested to the sample inlet, then add a quantitative amount of deionized water to rinse the sample inlet and the sample flow channel, drive the screening chip to centrifuge at a speed of R0 for 10s, and then rotate clockwise and counterclockwise alternately at a speed not greater than R0 to make the detection system in the detection chamber mix evenly. The reciprocating rotation program is as follows: clockwise rotation for 5 seconds at 100 rpm, counterclockwise rotation for 5 seconds at 100 rpm; repeat 10 times. S4. Incubate the screening chip at 37°C for 1.5 hours; S5. Centrifuge at R2 speed for 30s to allow the liquid in the detection chamber to break through the second flow channel and enter the waste liquid chamber. Then add deionized water to the sample inlet, centrifuge at R2 speed for 30s to rinse the detection chamber, and repeat the rinsing 3 times; Where R0 < R1 < R2; S6. Irradiate the detection cavity of the screening chip under 328nm excitation light, and detect the fluorescence intensity at 472nm and 633nm, respectively, and record them as F. 472 F 633 Calculate the ratio K, K=F 472 / F 633 Then, based on a pre-established standard curve characterizing the relationship between the value of K and NAD+ concentration... f The NAD+ concentration was analyzed, and the anticancer efficacy of the drug was determined based on the NAD+ concentration. The smaller the value of K, the lower the NAD+ concentration, indicating that the drug being tested has a better anticancer effect.

8. The drug screening method according to claim 7, characterized in that, The standard curve f It is constructed using the following method: 1) Add the test reagent to deionized water and ultrasonically disperse for 5-30 minutes to prepare a test reagent dispersion with a concentration of 0.1-2 mg / mL; 2) Add different amounts of NAD+ to the detection reagent dispersion, stir evenly, and prepare a series of standard working solutions with the same detection reagent concentration but different NAD+ concentrations. The NAD+ concentration range in this series of standard working solutions is 0-5 μmol / L. 3) Place the standard working solution at 37℃ and let it stand for 1-4 hours. Centrifuge and discard the centrifuged liquid. Add the obtained solid to deionized water to prepare a dispersion. Irradiate the dispersion under an excitation light of 328 nm and detect the fluorescence intensity at 472 nm and 633 nm, respectively, and record them as F. 472 F 633 Calculate the ratio K, K=F 472 / F 633 , with K B Using the x-axis as the horizontal axis and the NAD+ concentration as the y-axis, a curve fitting was performed to obtain the standard curve. f .

9. The drug screening method according to claim 7, characterized in that, R0=80-350rpm, R1=500-1750rpm, R2=1800-3000rpm.

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