Digital concentration readout of single enzyme molecules using femtoliter arrays and Poisson statistics.
Methods for accurately quantifying the concentration of a particular analyte in solution are all based on ensemble responses in which many analyte molecules give rise to the measured signal. In this paper, single molecules of beta-galactosidase were monitored using a 1 mm diameter fiber optic bundle with 2.4 x 10(5) individually sealed, femtoliter microwell reactors. By observation of the buildup of fluorescent products from single enzyme molecule catalysis over the array of reaction vessels and by application of a Poisson statistical analysis, a digital concentration readout was obtained. This approach should prove useful for single molecule enzymology and ultrasensitive bioassays. More generally, the ability to determine concentration by counting individual molecules offers a new approach to analysis of dilute solutions.
Rissin
David M
DM
Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, Massachusetts 02155, USA.
Walt
David R
DR
D001681:Biological Assay; D005336:Fiber Optic Technology; D007201:Indicator Dilution Techniques; D008832:Microchemistry / Q000379:methods*; D055100:Optical Fibers; D016012:Poisson Distribution; D040081:Protein Array Analysis; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism
D001616:beta-Galactosidase