Investigations On The Separation And Preconcentration Of Nucleic Acids And Proteins With Extraction By Ionic Liquids

The analysis of deoxyribonucleic acid (DNA) and proteins play a very important role in life science investigations and clinic diagnoses. In real world biological samples, DNA and proteins coexist with sample matrix components, thus, the separation of DNA and protein species of interest from the sample matrix is frequently called for. In conventional extraction procedures, the use of volatile and toxic organic solvents not only poses environmental problems, but also exhibit toxicity to DNA and protein species. At this point, it is most desirable to develop environmentally benign and green separation protocols. In this respect, ionic liquid as a kind of green solvent provides a promising alternative for the extraction and separation of biomolecules attributed to their distinct features of good heat stability, negligible vapor pressure and virtually no volatility.The present thesis focuses on the use of ionic liquid for developing green procedures for the extraction/separation of DNA and protein species. In addition, the interactions between the ionic liquid moiety and the biomolecules were exploited. More details are summarized in the following:(1) Double stranded (ds) DNA was effectively extracted in the absence of co-extractant or additive by using hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF6). An extraction efficiency of ca 95% could readily be obtained when 700?l DNA aqueous solution of 10 ng?l-1 was extracted by 200?l ionic liquid for 10 min at pH 7. A back extraction efficiency of ca 30% was achieved by using phosphate-citrate buffer. The extraction mechanisms were proposed and verified by 31P NMR and FT-IR spectra, i.e., interactions between cationic 1-butyl-3-methylimidazolium (Bmim+) and P-O bonds of phosphate groups in the DNA strands take place both in BmimPF6 dissolved in aqueous phase and at the interface of the two phases. The formation of DNA-Bmim adduct facilitates the transfer of DNA into the ionic liquid phase.(2) An abnormal resonance light scattering (RLS) arising from ionic liquid/DNA/EB (Ethidium Bromide) interactions was observed in ionic liquid phase and a desrease of RLS intensity of the DNA-EB system was observed with the increment of DNA concentration within a certain range. A quantification procedure for DNA in ionic liquid phase was thus developed based on this observation. Investigations of the mechanisms indicated that when dsDNA was extracted into ionic liquid the cationic Bmim+ groups of BmimPF6 intercalate into the DNA helix structure, which resulted in a reduction of the base-pair interstice along with transformation of DNA conformations that consequently prohibits the intercalation of EB with DNA. Thus, in the IL phase, the interactions between ethidium and DNA were dominated by electrostatic interactions and hydrogen bonding, leading to a congregation of EB entities around the DNA strands that results in an increase of absorption by ethidium, and consequently the inner filter effect leads to a reduction of the RLS.(3) Three hydrophobic imidazolium ionic liquids were prepared and employed to extract heme-protein species. It indicated that hemoglobin could be directly extracted into ionic liquid 1-butyl-3-trimethylsilylimidazolium hexafluorophosphate (BtmsimPF6) without using any co-extractant or additive; however, other protein species including bovine serum albumin (BSA), Transferrin, Cytochrome c remain in the aqueous solution. An extraction efficiency of ca 95% was obtained when 3 ml hemoglobin solution of 100 ng?l-1 was extracted by 400?l ionic liquid BtmsimPF6 for 30 min at pH 6.7. A back extraction efficiency of ca.85% for 20 ng?l-1 hemoglobin in ionic liquid phase was achieved with 0.6% sodium dodecyl sulfate (SDS) solution as stripping reagent.57Fe Mossbauer spectra and circular dichroism (CD) spectra indicated that the penta-coordinated ferrous atom in hemoglobin provide a vacant or free coordinating position, which could be occupied by the cationic Btmsim+ moiety. The interaction/coordination reaction between the iron atom in the heme group of hemoglobin and the cationic ionic liquid moiety furnishes the driving force for facilitating fast transfer of hemoglobin into BtmsimPF6.(4) A novel method for the extraction of cytochrome c (cyt-c) from aqueous solution into ionic liquid BtmsimPF6 was described. The results indicated that cyt-c could be extracted into ionic liquid BtmsimPF6 in an acidic medium. An extraction efficiency of ca.85% was achieved when extracting 3 ml cyt-c aqueous solution of 5.0 ng?l-1 with 400?l of BtmsimPF6 for 30 min at pH 1.0. A back extraction efficiency of ca 33% was obtained by using deionized water when 20 ng?l-1 cyt-c in 3 ml aqueous solution was extracted into ionic liquid. The extraction mechanisms were investigated by UV-Vis and CD spectra. It was demonstrated that conformation change of cyt-c was encountered in an acidic medium (pH<5) accompanied by the unfolding of peptide chain and the exposure of hydrophobic groups of heme, which results in the dissolution of cyt-c in ionic liquid BtmsimPF6. Meanwhile, a cleavage of the 6th coordination bond of the iron atom occurred at pH 1.0 by releasing the Met-80 ligand and giving rise to a vacant coordination position. Thus, the covalent coordination between the cationic Btmsim+ of ionic liquid and the iron atom of heme group facilitates the transfer of cyt-c into ionic liquid BtmsimPF6.(5) We have demonstrated that imidazolium ionic liquids emit strong fluorescence, and the fluorescence intensity was increased with the increment of the alkyl chain length of the cationic moiety, polarity of the solvent and pH of the system. Further investigations indicated that hemoglobin could effectively quench the fluorescence of the ionic liquid. This observation illustrated that imidazolium ionic liquids serve as a fluorescent probe for the assay of hemoglobin. A linear calibration curve was obtained in the range of 15-100 ng?l-1 with a detection limit of 5.0 ng?l-1 and a RSD of 3.2%(50 ng?l-1, n=11). The mechanisms of the present observation might be that the formation of a coordinating bond between the iron atom in the heme group of hemoglobin and the cationic moiety of the ionic liquids resulted in a decrease of the fluorescence of the ionic liquids. The fluorescence quenching in the present case might be attributed to static state quenching and energy transfer quenching.