A facile approach has been developed to synthesize Fe3O4@PAM（polyacrylamide） nanoparticles（NPs） with carboxyl groups on the surfaces by copolymerization with acrylamide and acrylic acid in Fe3O4 NPs aqueous suspen...A facile approach has been developed to synthesize Fe3O4@PAM（polyacrylamide） nanoparticles（NPs） with carboxyl groups on the surfaces by copolymerization with acrylamide and acrylic acid in Fe3O4 NPs aqueous suspension. Nitrilotriacetic acid（NTA） was conjugated to the magnetic NPs via well-known carboniimide chemistry using EDC and NHS. The Ni^（2＋） ions loaded on the surface of NPs provide abundant docking sites for immobilization of His-tagged green fluorescent proteins（His-tagged GFP）. The high magnetic property of Fe3O4@PAM@NTA-Ni^（2＋） allows an easy separation of the NPs from solution under an external magnetic field, with high His-tagged protein binding capacity（42 μg protein/mg of NPs）. The NPs can be recycled for at least four times without significant loss of binding capacity to proteins. These materials show great potential to separate His-tagged protein with low-cost purification at industrial scale.展开更多
Porous silicon(PSi)was applied as a supporting substrate for stepwise covalent derivatization of undecylenic acid, N-hydroxysuccinimidyl ester(NHS-ester)and nitrilotriacetic acid(NTA).By taking the advantages of porou...Porous silicon(PSi)was applied as a supporting substrate for stepwise covalent derivatization of undecylenic acid, N-hydroxysuccinimidyl ester(NHS-ester)and nitrilotriacetic acid(NTA).By taking the advantages of porous silicon as a supporting matrix such as high surface area to volume ratio,infrared transparency,porous semiconductors for laser desorption/ionization mass spectroscopy,and low fluorescence background,a multi-mode detection biochip prototype can be realized. We prepared such a protein microarray by spotting NTA microarray dots on NHS-ester derivatized PSi,converting the rest of chip area into poly(ethylene glycol)background,loading NiII,and finally affinity-binding histidine-tagged(His-tagged)proteins.With the multi-mode analyses of infrared spectroscopy,X-ray photoelectron spectroscopy(XPS),atomic force microscopy(AFM),matrix-assisted laser desorption/ionization mass spectroscopy(MALDI-MS),and fluorescence scanning,two example proteins,His-tagged thioredoxin-urodilatin and His-tagged aprotinin,were well qualified and quantified.展开更多
For many biomedical and catalytic applications, such as encapsulation of proteins/enzymes in nanopartides （NPs）, it is preferable to have well-dispersed small NPs that are stable in solution and behave quasi-homogen...For many biomedical and catalytic applications, such as encapsulation of proteins/enzymes in nanopartides （NPs）, it is preferable to have well-dispersed small NPs that are stable in solution and behave quasi-homogeneously. However, conventional liquid phase methods for small-NP synthesis and functionalization usually face great difficulties in separation/purification and recycling. In addition, controlling the orientation of proteins inside NPs is also a crucial issue to maximize the activity of the encapsulated proteins. Herein, we report a solid phase method to solve these problems. Using His-tagged proteins as cores, well-dispersed core-sheU silica NPs are facilely synthesized and functionalized in a column. The core His-tagged proteins are kept bound on the surface of the resIn beads in the column during the entire process, making the separation/purification of NPs and their precursors during the multiple-step process as simple as a few-minutes procedure of draining and washing the column. Each obtained silica NP has an adjustable eccentric core-shell structure with only one His-tag sticking out of the particle. This single His-tag on the surface of each NP not only makes it easy for addressable and stoichiometric functionalization of the NP but also provides an easy way to reversibly assemble NPs into dimers or be oriented on the surface of large particles. Notably, this solid phase approach also provides a versatile means to control the orientation of proteins inside NPs, and the His-tag makes it easy to recycle those well-dispersed small NPs.展开更多
基金Funded by the National Natural Science Foundation of China(Nos.21401051 and 51303049)Hubei Province Natural Science Foundation of China(Nos.2014CFB595 and 2014CFA080)+1 种基金Chutian Scholars Fund Project from the Education Department of Hubei ProvinceHundred Talents Program from the Organization Department of Hubei Province
文摘A facile approach has been developed to synthesize Fe3O4@PAM（polyacrylamide） nanoparticles（NPs） with carboxyl groups on the surfaces by copolymerization with acrylamide and acrylic acid in Fe3O4 NPs aqueous suspension. Nitrilotriacetic acid（NTA） was conjugated to the magnetic NPs via well-known carboniimide chemistry using EDC and NHS. The Ni^（2＋） ions loaded on the surface of NPs provide abundant docking sites for immobilization of His-tagged green fluorescent proteins（His-tagged GFP）. The high magnetic property of Fe3O4@PAM@NTA-Ni^（2＋） allows an easy separation of the NPs from solution under an external magnetic field, with high His-tagged protein binding capacity（42 μg protein/mg of NPs）. The NPs can be recycled for at least four times without significant loss of binding capacity to proteins. These materials show great potential to separate His-tagged protein with low-cost purification at industrial scale.
基金the financial support of the National Basic Research Program of China(2007CB925101)the National Natural Science Foundation of China(20721002&20827001)an open research fund of State Key Laboratory of Bioelectronics,Southeast University
文摘Porous silicon(PSi)was applied as a supporting substrate for stepwise covalent derivatization of undecylenic acid, N-hydroxysuccinimidyl ester(NHS-ester)and nitrilotriacetic acid(NTA).By taking the advantages of porous silicon as a supporting matrix such as high surface area to volume ratio,infrared transparency,porous semiconductors for laser desorption/ionization mass spectroscopy,and low fluorescence background,a multi-mode detection biochip prototype can be realized. We prepared such a protein microarray by spotting NTA microarray dots on NHS-ester derivatized PSi,converting the rest of chip area into poly(ethylene glycol)background,loading NiII,and finally affinity-binding histidine-tagged(His-tagged)proteins.With the multi-mode analyses of infrared spectroscopy,X-ray photoelectron spectroscopy(XPS),atomic force microscopy(AFM),matrix-assisted laser desorption/ionization mass spectroscopy(MALDI-MS),and fluorescence scanning,two example proteins,His-tagged thioredoxin-urodilatin and His-tagged aprotinin,were well qualified and quantified.
基金This work was supported by the National Natural Science Foundation of China （NSFC） （Nos. 21371117 and 31571024） and the National Basic Research Program of China （No. 2016YFA0201602）.
文摘For many biomedical and catalytic applications, such as encapsulation of proteins/enzymes in nanopartides （NPs）, it is preferable to have well-dispersed small NPs that are stable in solution and behave quasi-homogeneously. However, conventional liquid phase methods for small-NP synthesis and functionalization usually face great difficulties in separation/purification and recycling. In addition, controlling the orientation of proteins inside NPs is also a crucial issue to maximize the activity of the encapsulated proteins. Herein, we report a solid phase method to solve these problems. Using His-tagged proteins as cores, well-dispersed core-sheU silica NPs are facilely synthesized and functionalized in a column. The core His-tagged proteins are kept bound on the surface of the resIn beads in the column during the entire process, making the separation/purification of NPs and their precursors during the multiple-step process as simple as a few-minutes procedure of draining and washing the column. Each obtained silica NP has an adjustable eccentric core-shell structure with only one His-tag sticking out of the particle. This single His-tag on the surface of each NP not only makes it easy for addressable and stoichiometric functionalization of the NP but also provides an easy way to reversibly assemble NPs into dimers or be oriented on the surface of large particles. Notably, this solid phase approach also provides a versatile means to control the orientation of proteins inside NPs, and the His-tag makes it easy to recycle those well-dispersed small NPs.
