Protein assembly onto patterned microfabricated devices through enzymatic activation of fusion pro-tag.
We report a versatile approach for covalent surface-assembly of proteins onto selected electrode patterns of pre-fabricated devices. Our approach is based on electro-assembly of the aminopolysaccharide chitosan scaffold as a stable thin film onto patterned conductive surfaces of the device, which is followed by covalent assembly of the target protein onto the scaffold surface upon enzymatic activation of the protein's "pro-tag." For our demonstration, the model target protein is green fluorescent protein (GFP) genetically fused with a pentatyrosine pro-tag at its C-terminus, which assembles onto both two-dimensional chips and within fully packaged microfluidic devices in situ and under flow. Our surface-assembly approach enables spatial selectivity and orientational control under mild experimental conditions. We believe that our integrated approach harnessing genetic manipulation, in situ enzymatic activation, and electro-assembly makes it advantageous for a wide variety of bioMEMS and biosensing applications that require facile "biofunctionalization" of microfabricated devices.
Lewandowski
Angela T
AT
Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland, USA.
Yi
Hyunmin
H
Luo
Xiaolong
X
Payne
Gregory F
GF
Ghodssi
Reza
R
Rubloff
Gary W
GW
Bentley
William E
WE
D001665:Binding Sites; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
D011993:Recombinant Fusion Proteins; D014442:Monophenol Monooxygenase