Femtoliter-scale separation and sensitive detection of nonfluorescent samples in an extended-nano fluidic device

The separation and sensitive detection of nonfluorescent molecules at the femtoliter (fL) scale has been achieved for the first time in a nanofluidic channel. Smaller sample volumes and higher separation efficiencies have been significant targets for liquid chromatography for many years. However, the use of packed columns hindered further miniaturization and improvement of separation efficiency. Our group recently developed a novel chromatographic method using an open nanofluidic channel to realize attoliter sample injection and a separation efficiency of several million plates per m. However, because of the extremely small optical path length, this detection method was limited to fluorescent molecules. Herein, we describe the combination of nanofluidic chromatography with differential interference contrast thermal lens microscopy (DIC-TLM), a sensitive detection method for nonfluorescent molecules developed by our group that has the ability to detect 0.61 zmol (370 molecules) with an optical path length of 350 nm. As a result, separation of a 21 fL sample containing 250 zmol was possible at the limit of detection (LOD). Liquid chromatography using a nanofluidic chip and DIC-TLM realized separation and detection of a 21 fL, 0.61 zmol nonfluorescent sample.