| Drug screening plays a vital role in the process of new drug research and development,and high-throughput drug screening can speed up the process of drug research and development.As a new technology,microfluidic chip can integrate several traditional operating units in the size of several millimeters or even microns,and has the advantages of high integration,high throughput,low consumption,low cost and automation.It is very suitable to be used as a drug screening platform.In this paper,firstly,the traditional drug screening method was used to isolate and identify the anti-tumor active components in the seeds of Macleaya cordata.Finally,according to the laminar flow and microdialysis theory of microfluidic chips,two new types of microfluidic chips were established for multi-target competitive affinity screening.the main contents are as follows:The first chapter is the preface,which describes the research progress of drug screening and the research status of traditional Chinese medicine active components screening,and analyzes the drug screening methods and applications based on microfluidic chip in detail.In the second chapter,the contrast affinity ultrafiltration method was established,and the components of the filtrate after ultrafiltration were detected by HPLC-MS,and compared with the blank control,the ligands that could bind to human telomere DNA(HT24)in Macleaya tomentosa seeds were screened.The results showed that the relative abundance was more than 30% and the difference of peak area was more than 20%.The circular dichroism conformation of sanguinine(SAN)and chelerythrine(CHE),were identified by mass spectrometry.it was further proved that SAN and CHE were active ligands of HT4.In addition,protopine(PRO)and allocryptopine(ALL),which have large abundance but little difference in peak area,were identified.The binding rate of SAN,CHE,PRO,ALL was calculated and compared by molecular docking method.In the third chapter,a new three-phase laminar flow chip was constructed to realize the competition of multi-components against double targets(G-quadruplex,HT24;double-stranded DNA,DNA26)in the extract of traditional Chinese medicine,in order to screen the anti-tumor components with strong targeting.The chip is equipped with a guide structure and isolated at the bend to keep the laminar flow more stable,the two biological targets do not interfere with each other,and the drug small molecules combine competitively with the two targets by diffusion.The results showed that there were ten kinds of compounds that could bind to two kinds of targets,among which there were five compounds with no significant difference in binding to double-stranded DNA and G4,and five compounds with obvious binding differences.The binding constants and binding modes of the four alkaloids with two targets were verified by fluorescence spectroscopy and molecular docking methods.It is proved that sanguinarine and chelerythrine can be used as drug candidates with good targeting.In chapter 4,a 3D array chip was constructed to realize the competitive screening of four different biological targets(enzymes: DNA topoisomerase Ⅱ α and DNA topoisomerase Ⅱ β;DNA:HT24 and DNA26).Four different targets are isolated by porous membranes and independent target channels,and drug molecules can freely pass through the porous membranes to compete with different targets.Among them,the results of DNA26 and HT24 are basically consistent with those of chapters 2 and 3,indicating that the 3D array chip is reliable and can be used for multi-target to multi-component drug screening.The results of screening were as follows: the binding degree of CHE: HT24 > TOP2 B > DNA26 > TOP2A;SAN: TOP2 A > TOP2 B > HT24 > DNA26;ALL: HT24 > TOP2 B >DNA26 > TOP2A;PRO: HT24 > TOP2 B > TOP2 A > DNA26.Finally,the binding mode and efficacy of the selected molecules were analyzed by molecular docking and cytotoxicity experiments,and it was found that CHE had more potential to become a medicine than SAN. |
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