Synthesis Of Biofunctionalized Micro/Nano Materials And Their Application In Affinity Separation Of Proteins

Three kinds of inorganic micro/nano materials including SiO₂, FeOOH, and Fe₃O₄were selected assupporters and biologically surface functionalized. Resultant biologically surface functionalized SiO₂,FeOOH, and Fe₃O₄were then adopted to separate and purify glutathione S-transferase （GST） and fusionproteins tagged by hexahistidine （denoted as His-tagged proteins）. The morphology, structure, andperformance of various as-fabricated biologically surface functionalized inorganic micro/nano materialswere analyzed using various modern analytical techniques, and their ability for affinity separation ofproteins was evaluated. The main scopes and results of this thesis are as follows.（1） Preparation and biological functionalization of nanometer silica as well as its application inseparation of GSTDumbbell-, fiber-and chain-like nanosized silicas were synthesized by hydrolysis of tetraethylorthosilicate （TEOS）. The influences of the mode for introducing TEOS and the concentration of ammoniaand TEOS on the morphology of nanosized silicas were studied, and the possible formation mechanism ofvarious nanosized silicas was discussed. As-prepared silica nanospheres were then capped with amino,aldehyde and glutathione groups to afford SiO₂-GSH microspheres with unique adsorption capabilitytowards GST. The morphology and structure of as-synthesized SiO₂-GSH microspheres and their ability toisolate proteins were analyzed and evaluated by scanning electron microscopy （SEM）, ultraviolet-visiblespectrophotometry （Uv-vis）, thermogravimetric analysis （TG） and sodium dodecylsulfate-polyacrylamidegel electrophoresis （SDS-PAGE）. Results indicate that SiO₂-GSH microspheres are suitable for the initialgross separation and purification of GST.（2） Preparation of thiol-silica microspheres and their application in separation of GSTThiol-containing silica nanosized microspheres （nSiO₂-SH） were synthesized via one-step hydrolysisof （3-mercaptopropyl）trimethoxysilane （MPS）, and the effects of water to ethanol ratio, reactiontemperature, time and MPS amount on the morphology of resulting products were investigated. Possiblereaction mechanism was proposed. Moreover, the content of surface thiol group （―SH） of nSiO₂-SHmicrospheres was measured by DTNB method and X-ray photoelectron spectroscopy, and it was found that the content of surface thiol group of nSiO₂-SH microspheres is strongly dependent on the reactionconditions. Furthermore, as-prepared nSiO₂-SH microspheres were allowed to react with glutathionethrough disulfide bond （-S-S-） to produce biologically functionalized nSiO₂-GSH microspheres. Themorphology, size and chemical structure of as-obtained nSiO₂-GSH microspheres were characterized byscanning electron microscopy, transmission electron microscopy, and Fourier transform infraredspectrometry. Their thermal stability was evaluated by thermogravimetric analysis, and their ability tocapture and isolate GST was detected by SDS-PAGE method. Results show that GST can be specificallyadsorbed on nSiO₂-GSH microspheres in the mixed protein solution. Therefore, nSiO₂-GSH microspherehas potential applications in the separation, purification and detection of GST-tagged fusion proteins.（3） Synthesis of petal-like ferric oxide/cysteine architectures and their application in separation ofHis-tagged proteinsMicro/nano composites of petal-like ferric oxide functionalized by L-cysteine （FeOOH/Cys） wereprepared through a one-step solvothermal route. Ni²⁺was then adsorbed onto as-synthesized FeOOH/Cysmicro/nano composites which were adopted to enrich and isolate His-tagged proteins directly from themixture of lysed cells without sample pretreatment. The effects of initial mass of FeOOH/Cys and amountof E. coli lysate on the isolation efficiency of His-tagged proteins were investigated. Results show thatFeOOH/Cys micro/nano composites with immobilized Ni²⁺possess good ability to isolate His-taggedOST1proteins and exhibit a saturation capacity of88.0μg/mg. Besides, they exhibit good recyclability andcan still effectively isolate target proteins after four cycle of regeneration.（4） Synthesis of Fe₃O₄/Cys microspheres and their application in purification of His-tagged proteinsMagentic microspheres of nanosized Fe₃O₄functionalized by L-cysteine （denoted as Fe₃O₄/Cys） werefabricated via a solvothermal route. Resultant Fe₃O₄/Cys microspheres were then allowed to adsorb Ni²⁺affording Fe₃O₄/Cys-Ni²⁺composite microspheres. As-obtained Fe₃O₄/Cys-Ni²⁺microspheres were used toenrich and separate His-tagged proteins directly from the mixture of lysed cells without samplepretreatment. Results show that Fe₃O₄/Cys-Ni²⁺microspheres possess good ability to isolate His-taggedTRX proteins and exhibit a saturation adsorption capacity of53.2μg/mg. Besides, they exhibit goodrecyclability and retain good isolation performance towards His-tagged proteins even after multi-cycle ofregeneration.