Single-molecule mobility and spectral measurements in submicrometer fluidic channels

Electrophoretic mobility differences of biological molecules are frequently exploited to physically separate and subsequently identify the components of a mixture. We present a method to rapidly identify single molecules by measuring both their mobility and fluorescence emission under continuous flow without separation. Submicrometer fluidic channels were used to detect individual nucleic-acid-engineered fluorescent labels driven electrokinetically in free solution. Two separate focal volumes along the length of the fluidic channel collected spectral, spatial, and temporal information from the passage of fluorescent labels through the channel. One focal volume was defined by a focused 488-nm -wavelength laser and the other by a focused 568-nm laser. The subfemtoliter focal volumes resulted in signal-to-noise ratios sufficient for single-fluorophore detection, and the two excitation wavelengths enabled detection of multicolor fluorescent labels and discrimination of single-color detection events. Each fluorescent label was uniformly excited and analyzed as it passed through the channel. Flow control facilitated high throughput at low concentrations, as well as a balance of several experimental parameters. Two fluorescent labels were considered for identification by single-molecule mobility measurements. Approximately 81% of fluorescent labels of one variety and 77% of the other were found to be identifiable based on their mobility alone. As implemented, this method could be used in conjunction with spectral analysis of fluorescent labels to enhance the resolution of single-molecule identification. Ultimately, this technique could be used autonomously to detect biomolecules bound to fluorescent labels and to identify similarly labeled biomolecules by their mobility.