Application Of Guanidinium Ionic Liquids For Traditional Chinese Medicine And Protein Extraction Research And Electromechanical Properties Research Of Cells

As a green solvent,ionic liquid has been widely concerned due to its unique physicochemical properties.Ionic liquid-aqueous two phase extraction is a new separation technique for bioactive compounds.Functional ionic liquid-aqueous two phase system has advantages of both ionic liquid and aqueous two phase extraction.It is non-toxic,safe,easy-to-use and fast.It has been widely used in biotransformation and purification of protein,nucleic acid and virus.Functional ionic liquid-magnetic solid phase extraction is a noval technique which has some certain function by modifying functional ionic liquid onto magnetic absorbent.Guanidinium ionic liquid is a new member of ionic liquid family.It can be easily prepared and has excellent thermal and chemical stability,strong biolog ical activity and great variability in designing new species.The first three works of my research were: synthesis of guanidinium ionic liquid and its application in traditional Chinese medicine Radix Peucedani extraction;and functionalization of guanidinium ionic liquid and its application of protein extraction by aqueous two phase extraction technique and magnetic solid phase extraction technique.Then,I was heavily involved in advanced research on electrophysiological properties of cardiomyocyte cell and mechani cal properties of cancer cell in 2014-2016 as a joint Ph D at UCLA.Our work was to know maturation process of cardiomyocytes at electrophysiological level,and to investigate the role of glucose in this maturation process.Also,we proposed a method to measure mechanical properties for bladder cancer cell at single cell level,not only can provide high throughput detection,but also have potential to be used in clinical application.The main work we carried out is as follows:(1)Guanidinium ionic liquid based microwave-assisted extraction for Praeruptorin A from Radix peucedaniThis chapter introduced a guanidinium ionic liquid based microwave-assisted extraction method for Praeruptorin A extraction from Radix peucedani.After extraction,reversed-phase high-performance liquid chromatography with UV detection was employed to analyze Praeruptorin A.Several significant experimental parameters were systematically optimized by single-factor and L9(34)orthogonal array methodology.The amount of Praeruptorin A extracted by [1,1,3,3-tetramethylguanidine]-CH2CH(OH)COOH is the highest,reaching 11.05 ± 0.13 mg/g.Guanidinium ionic liquid based microwave-assisted extraction presents unique advantages in Praeruptorin A extraction,comparing with guanidinium ionic liquid based maceration extraction,guanidinium ionic liqui d based heat reflux extraction and guanidinium ionic liquid based ultrasound-assisted extraction.Linearity study results showed good linearity of Pd-Ia in 0.005-0.200 mg/m L.The LOD was 2.07 ?g/m L.Recovery range of sample was 99.23-100.07%.Precision,repeatability and stability were all satisfactory.The RSD of precision experiment and repeatability experiment were 0.34% and 0.52%,respectively.The RSD of stability were 0.67% and 1.0%.The mechanisms of guanidinium ionic liquid based microwave-assisted extraction were studied by scanning electron microscopy and FTIR spectroscopy.This work showed that guanidinium ionic liquid based microwave-assisted extraction has a huge potential in the extraction of bioactive compounds from complex samples.(2)Functional guanidinium ionic liquid-aqueous two phase system for protein extractionThis chapter introduced a series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids.Functional guanidinium ionic liquid aqueous two-phase systems(FGIL-ATPSs)have been designed with these functional guanidinium ionic liquids and phosphate solution for the extraction of protein.Concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible spectrophotometer.Single factor experiments were used to investigate the effects of several parameters on the proposed process.The best extraction condition is: 4.0 mmol of ILs,0.6 g/m L of salt solution,25? of temperature and 12-24 mg of protein.The extraction efficiency reaches 97.05%.The advantages of FGIL-ATPSs such as stronger phase forming ability and higher extraction amoun t were demonstrated by comparison with regular ionic liquid aqueous two-phase systems.Precision,repeatability and stability were all satisfactory,the corresponding RSD were 1.1%,1.6% and 1.8%.The mechanisms of purification were researched by dynamic l ight scattering,determination of the conductivity and transmission electron microscopy.Structure of protein before and after extraction has been studied by using UV,FTIR and CD.The results showed structure of protein has not changed after extraction.This work shows that FGIL-ATPSs have huge potential to offer new possibilities in the purification of proteins.(3)Functional guanidinium ionic liquid modified magnetic particles for solid phase extraction of proteinThis chapter prepared some magnetic chitosan graphene oxide(MCGO)composite,functional guanidinium ionic liquids were coated on MCGO to extract protein by magnetic solid-phase extraction.The synthesized magnetic absorbent(MCGO-FGILs)has been characterized by FTIR,VSM,SEM and XRD.Concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible spectrophotometer.A comprehensive study on the adsorption conditions were conducted.The best extraction condition was: 2.5 mg/m L of protein concentration,12 mg of MCGO-FGILs,5.0 of p H,30? of temperature and 30 minutes of extraction time.Extraction amount of different kinds of proteins were as follows: 18.9833 mg/g(BSA),20.6725 mg/g(OVA),35.2640 mg/g(Try),38.4006 mg/g(Lys).The advantageous properties of MCGO-functional guanidinium ionic liquid in protein extraction were compared with magnetic chitosan,graphene oxide,MCGO and MCGO-ordinary imidazolium ionic liquid,extraction efficiency of MCGO-functional guanidinium ionic liquid is the highest,which reaches 48.8 mg/g.Moreover,the MCGO-functional guanidinium ionic liquid can be easily regenerated,its extraction capacity was about 94.0% of the initial one after being used three times.Method validation result showed RSD of precistion,repeatabilit y and stability were 1.3%,1.7% and 1.0%,respectively.(4)Electrophysiological characterization of the maturation process of mouse ESC-CMsThis chapter introduced a system for extracellular field potential monitoring that utilizes a two-dimensional confluent layer of mouse embryonic stem cells derived cardiomyocytes.Microelectrode array system has been used to investigat e electrophysiological signals of cells in maturation process.The signal is like electrocardiogram.After analyzing beating rate,field pot ential amplitude and field potential duration of the cells,we found the pace maker cells and conduction trend of electrical signal.The result showed,amplitude and conduction speed both increased in maturation process,but beat interval and field potenti al duration were stable.Beat interval was about 1.5 s,field potential duration was 0.2-0.4 s.This work allows understanding of the behavior of mouse ESC-CMs maturation.(5)Influence of glucose deprivation on h ESC-CMs maturationThis chapter introduced a system for extracellular field potential monitoring that utilizes a two-dimensional confluent layer of human embryonic stem cells derived cardiomyocytes.Microelectrode array system has been used to investigat e electrophysiological signals of cells in maturation process.In addition,influence of glucose deprivation on h ESC-CMs maturation has been researched.The signal was like electrocardiogram.The result showed that amplitude,conduction speed and d V/dt have increased from 420.6 ?V?31.9 mm/s and 2.8 ?V/ms to 609.8 ?V?89.5 mm/s and 17.2 ?V/ms,separately,under glucose deprivation condition.This work showed glucose deprivation can promote cardiomyocytes maturation.(6)Mechanical characterization of bladder cancer cellThis chapter introduced a mechanical characterization method for bladder cancer cells based on single cell level.Atomic force microscopy(AFM)has been used to research cell stiffness under 4-Aminobiphenyl.AFM tip could be accurately controlled.We calculated cells stiffness under different indentation.Matlab has been used for data analysis.Mechanical curve from AFM was fitting by Sneddon model for Young's modulus calculation and 2D maps.The result showed that Young 's modulus decreased from 100 k Pa to 10 k Pa as indentation increased from 100 nm to 1000 nm.This work lays a foundation of cells mechanical phenotype research and provides possibilities for further research of cells mechanical properties in molecular level.