Microfabricated arrays of femtoliter chambers allow single molecule enzymology.
Precise understanding of biological functions requires tools comparable in size to the basic components of life. Single molecule studies have revealed molecular behaviors usually hidden in the ensemble- and time-averaging of bulk experiments. Although most such approaches rely on sophisticated optical strategies to limit the detection volume, another attractive approach is to perform the assay inside very small containers. We have developed a silicone device presenting a large array of micrometer-sized cavities. We used it to tightly enclose volumes of solution, as low as femtoliters, over long periods of time. The microchip insures that the chambers are uniform and precisely positioned. We demonstrated the feasibility of our approach by measuring the activity of single molecules of beta-galactosidase and horseradish peroxidase. The approach should be of interest for many ultrasensitive bioassays at the single-molecule level.
Rondelez
Yannick
Y
Tresset
Guillaume
G
Tabata
Kazuhito V
KV
Arata
Hideyuki
H
Fujita
Hiroyuki
H
Takeuchi
Shoji
S
Noji
Hiroyuki
H
D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods; D006735:Horseradish Peroxidase / Q000032:analysis* / Q000737:chemistry; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D008904:Miniaturization; D008967:Molecular Biology / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D001616:beta-Galactosidase / Q000032:analysis* / Q000737:chemistry
D006735:Horseradish Peroxidase; D001616:beta-Galactosidase