| Deep eutectic solvent(DES),as a new kind of green solvent,has aroused considerable concern due to its excellent physical and chemical properties,synthetically accessible,and biodegradable.Aqueous two phase system(ATPS)is an efficient and temperate liquid-liquid separation technique.Aqueous two phase system based on deep eutectic solvent has the excellent properties of deep eutectic solvent and aqueous two phase system such as green,non-toxic and biocompatibility.Biomacromolecules are important biological substances,the separation and extraction of these biomolecules is of great significance in life analysis,clinical diagnosis and disease prevention.It is of great advantage to extract and separate biological macromolecules by deep eutectic solvent based aqueous two phase system,the extraction environment is close to the physiological environment of biological materials,after extraction the biological activity of biomacromolecules are remained.Moreover,the method has the advantages of high extraction efficiency,simple equipment operation,easy amplification and no residual organic solvent.In this research,the methods based on the deep eutectic solvent aqueous two phase system for the extraction of biomacromolecules such as protein,DNA,RNA,were established.The main work we carried out is as follows:(1)Betaine-based deep eutectic solvent aqueous two phase system for the extraction of proteinSix kinds of new type of green betaine-based deep eutectic solvent(DES)have been designed and synthesized.Deep eutectic solvent aqueous two phase systems(DES-ATPS)were established and successfully applied in the extraction of protein.Bovine serumal bumin(BSA)used as target protein,Betaine-urea(Be-U)was selected as the suitable extractant.Single factor experiments were carried out to determine the optimum conditions of the extraction process,such as the salt concentration,the mass of DES,the separation time,the amount of protein,the temperature and the pH value.The extraction efficiency could achieve to 99.82%under the optimum conditions.Mixed sample experiment and practical sample experiment show that the aqueous two phase system could extract the protein from the complex matrix.The back extraction experiment showed that the back extraction efficiency could reach to 32.66%.The precision,repeatability and stability experiments showed that precision of the instrument,reproducibility and stability of the method were excellent.UV-vis,FTIR and CD spectra confirmed that the conformation of protein was not changed during the process of extraction.The mechanisms of extraction were researched by DLS,the measurement of the conductivity and TEM,and the results indicated that DES-protein aggregates and embraces phenomenon play considerable roles in the separation process.The proposed method provides a potential method for the separation of proteins.(2)Tetrabutylammonium bromide-based deep eutectic solvent aqueous two phase systems for the extraction of DNAFour kinds of novel green deep eutectic solvent were designed and synthesized based on tetrabutylammonium bromide,ethylene glycol,propylene glycol,butylene glycol,and butyl alcohol,respectively.DES-based aqueous two phase systems were developed and applied in the rapid and efficient extraction of DNA from salmon testes.Tetrabutylammonium bromide-ethylene glycol and sodium sulfate were selected as the appropriate phase components.Single factor experiments proved that the extraction efficiency of DNA was influenced by the mass of DES,the salt concentration,the temperature,the separation time,the pH value,and the ionic strength value.After extraction,99.98%of the DNA was transferred to the DES-rich top phase.In order to meet the requirements of downstream applications and obtain high purity DNA,the back extraction experiments were carried out and 89.69%of the DNA could be back extracted into the salt-rich phase.Then the extraction and back-extraction were each applied to DNA complex samples containing protein,which proved that DNA could be selectively separated from complex samples.The precision,repeatability and stability experiments showed that precision of the instrument,reproducibility and stability of the method were excellent.FTIR spectra,CD spectra,DLS and TEM were combined to investigate the binding characteristics and the extraction mechanism,the results indicated that electrostatic interaction play considerable roles in the separation process.The proposed method provides a potential method for the selective separation of DNA.(3)Deep eutectic solvent based aqueous two phase systems for the extraction of RNAEight kinds of deep eutectic solvent synthesis were synthesized,the phase behavior between these deep eutectic solvents and four kinds of salt,the phase forming ability of this series of aqueous two phase systems were compared.Then the extraction capacity of the series of aqueous two phase systems of RNA were explored,TBAB/K2HPO4 was selected as the appropriate extraction system.Single factor experiments proved that the extraction efficiency of DNA was influenced by the mass of DES,the salt concentration,the temperature,the separation time,the pH value,and the ionic strength value,after optimization experiment the extraction efficiency of RNA was 94.78%.Then NaCl was used as the reverse extractant to investigate the effect of the different phase forming salt on the back extraction of RNA,the results showed that when NaH2PO4 was used as the phase forming salt,the reverse extraction efficiency of RNA was up to 86.55%.The experiments of mixed sample extraction were carried out,which demonstrated that the selective separation of RNA and protein could be realized.The precision,repeatability and stability experiments showed that precision of the instrument,reproducibility and stability of the method were excellent.The extraction mechanism of RNA was studied by conductivity,DLS and TEM,the results indicated that electrostatic interaction play considerable roles in the separation process.The proposed method provides a potential method for the selective separation of RNA. |
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