Preparation Of Hybrid Hydrogels And Application In Electrophoretic Separation Of Proteins

As a hydrogel matrix separation technology,gel electrophoresis is widely employed in molecular biology,genetics and biochemistry.Compared with other electrophoresis,the hydrogel not noly prevent the convection and reduce the diffusion,but also with the molecular sieve effect due to porous medium.Gel electrophoresis is used to separation and purification of the biosamples before the Mass spectrum(MS),Polymerase chain reaction(PCR)and Western blot(WB).The conventional gel matrix could only separation of proteins with molecular weight(Mw)range of 30~250kDa because of the single structure and poor performances,and this greatly limits the biological application of gel electrophoresis.Therefore,it is vital to develop simple synthesis method and high resolution composite hydrogel for biologic application.This thesis mainly focuses on the design and synthesis of the novel hybrid hydrogel as separation matrix for gel electrophoresis,and applied for bioseparation.The concrete research content is as follow:(1)Poly(acrylamide-acrylic acid)(P(AM-AA))hydrogel was prepared via one-pot chemical copolymerization in a slit between two glass plates(1.0×70×100 mm~3).The morphology phenomenon and the electrophoretic separation effect were studied using gel electrophoresis,Mass spectrum(MS),scanning electron microscope(SEM),Fourier Transforminfraredspectroscopy(FTIR),frequency sweeping,Thermogravimetric analyses(TGA)and Differential scanning calorimetry(DSC).Further study showed that the pores size of P(AM-AA)hydrogel was smaller than that of the Polyacrylamide hydrogel obviously.The crosslink density of the P(AM-AA)hydrogel was increased due to the multiple crosslinked between acrylamide and acrylic acid,and the mechanical properties was similar to the polyacrylamide(PAM)hydrogel.Gel electrophoresis indicated that the multiple covalent crosslinked hydrogel exhibited high resolution separation by copolymerization low concentration of acrylamide and acrylic acid.Especially,the resultant hydrogel exhibited high separation performance of proteins with Mw more than 250 kDa.Several ultrahigh Mw proteins,such as DMBT1(268 kDa),DESP(334 kDa),MUC5B(611 kDa)could be successfully separated from the human saliva through the P(AM-AA)hydrogel.The formation mechanisms were discussed and the architecture of the resultant hydrogel was proposed.(2)Poly(acrylamide-co-acrylic acid)(P(AM-co-AA))hydrogel was synthesized via one-pot copolymerization in a slit between two glass plates(1.0×70×100 mm~3).Gel electrophoresis indicated that the chemical and physical crosslinked hydrogel exhibited high resolution separation by copolymerization acrylamide,acrylic acid and calcium chlordie.Especially,the hybrid hydrogel exhibited high separation performance of proteins with Mw less than 30 kDa.Several low Mw proteins,such as IGLL5(23.4 kDa),ZG16B(22.7 kDa),LCN1(19.4 kDa),LC1L1(18 kDa),PIP(16.8kDa),IGKC(11.8 kDa)and LAC2(11.5 kDa)could be successfully separated from the human saliva using the P(AM-co-AA)hydrogel.The morphology phenomenon and the electrophoretic separation effect were studied using gel electrophoresis,Mass spectrum(MS),scanning electron microscope(SEM),Fourier Transform infrared spectroscopy(FTIR),frequency sweeping,Thermogravimetric analyses(TGA)and Differential scanning calorimetry(DSC).Further study showed that the pores size of P(AM-co-AA)hydrogel not only smaller but also regular than that of the PAM hydrogel.The crosslink density of the P(AM-co-AA)hydrogel was increased due to the ionic coordination between Ca(II)and carboxyl group,meanwhile the multi covalent crosslinked among acrylamide,acrylic acid and N,N’-methylene bis-acryclamide(MBA).Double crosslinked could improve the mechanical properties of the P(AM-co-AA)hydrogel.The formation mechanisms were discussed and the architecture of the hybrid hydrogel was proposed.(3)Antimicrobial peptide could be rapidly and successfully separation via the P(AM-co-AA)hydrogel.Because of compatible with the current experimental techniques,laboratory apparatus and chemical reagents,the hybrid hydrogel has very fine application prospects in the fields of biological research and clinical diagnosis.