Real-time monitoring of enzyme activity in a mesoporous silicon double layer.
The activity of certain proteolytic enzymes is often an indicator of disease states such as cancer, stroke and neurodegeneracy, so there is a need for rapid assays that can characterize the kinetics and substrate specificity of enzymatic reactions. Nanostructured membranes can efficiently separate biomolecules, but coupling a sensitive detection method to such a membrane remains difficult. Here, we demonstrate a single mesoporous nanoreactor that can isolate and quantify in real time the reaction products of proteases. The reactor consists of two layers of porous films electrochemically prepared from crystalline silicon. The upper layer, with large pore sizes ( approximately 100 nm in diameter), traps the protease and acts as the reactor. The lower layer, with smaller pore sizes ( approximately 6 nm), excludes the proteases and other large proteins and captures the reaction products. Infiltration of the digested fragments into the lower layer produces a measurable change in optical reflectivity, and this allows label-free quantification of enzyme kinetics in real time within a volume of approximately 5 nl.
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, Department 0358, La Jolla, California 92093-0358, USA.
Manuel M
MM
Orosco
Claudia
C
Pacholski
Michael J
MJ
Sailor
D003199:Computer Systems; D003460:Crystallization / Q000379:methods*; D004789:Enzyme Activation; D004798:Enzymes / Q000032:analysis* / Q000737:chemistry*; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry; D008422:Materials Testing; D008567:Membranes, Artificial; D008968:Molecular Conformation; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure; D036103:Nanotechnology / Q000379:methods*; D010316:Particle Size; D016062:Porosity; D012825:Silicon / Q000737:chemistry*; D013499:Surface Properties
D004798:Enzymes; D046911:Macromolecular Substances; D008567:Membranes, Artificial; D012825:Silicon