Oil-sealed femtoliter fiber-optic arrays for single molecule analysis.
This paper describes a novel method for fabricating and sealing high-density arrays of femtoliter reaction chambers. We chemically etch one end of a 2.3 mm diameter glass optical fiber bundle to create an array of microwells. We then use a contact printing method to selectively modify the surface of the material between microwells with a hydrophobic silane. This modification makes it possible to fill the wells with aqueous solution and then seal them with a droplet of oil, forming an array of isolated reaction chambers. Individual β-galactosidase molecules trapped in these reaction chambers convert a substrate into a fluorescent product that can be readily detected because a high local concentration of product is achieved. This binary readout can be used for ultra-sensitive measurements of enzyme concentration. We observed that the percentage of wells showing enzyme activity was linearly dependent on the concentration of soluble β-galactosidase in the picomolar range. A similar response was also observed for streptavidin-β-galactosidase captured by biotinylated beads. These arrays are also suitable for performing single-molecule kinetics studies on hundreds to thousands of enzyme molecules simultaneously. We observed a broad distribution of catalytic rates for individual β-galactosidase molecules trapped in the microwells, in agreement with previous studies using similar arrays that were mechanically sealed. We have further demonstrated that this femtoliter fiber-optic array can be integrated into a PDMS microfluidic channel system and sealed with oil on-chip, creating an easy to use and high-throughput device for single-molecule analysis.
Department of Chemistry, Tufts University, Medford, Massachusetts, USA.
Huaibin
H
Zhang
Shuai
S
Nie
Candice M
CM
Etson
Raymond M
RM
Wang
David R
DR
Walt
D005336:Fiber Optic Technology; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D046210:Microfluidic Analytical Techniques; D009821:Oils / Q000737:chemistry*; D040081:Protein Array Analysis; D012821:Silanes / Q000737:chemistry; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry; D001616:beta-Galactosidase / Q000032:analysis
D005456:Fluorescent Dyes; D009821:Oils; D012821:Silanes; D014867:Water; D001616:beta-Galactosidase