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

Abstract

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).

Femtoliter high-performance liquid chromatography using extended-nano channels

“…7 From this result, we calculated the injection volume using the fluorescence intensity and peak area as reported previously. 17 The estimated injection volume was 35 fL, which was extremely small compared to the nL volume achieved by conventional nanoflow LCs. However, the volume was 1-2 orders larger than the previous report.…”

Accelerated protein digestion and separation with picoliter volume utilizing nanofluidics

“…The power of the excitation and the probe beams were 7.8 mW and 27 mW under the objective lens, respectively. The excitation beam was modulated using a mechanical chopper with a modulation frequency at 1.2 kHz, and the time constant of the lock-in amplifier was 300 ms. 46 After finishing the measurement, 100 mM borate buffer was introduced at the applied pressure of P 3 = 500 kPa for over 10 min. Then, the valve was opened.…”

Differential interference contrast‐photothermal microscopy in nanospace: impacts of systematic parameters

“…DIC‐PTM shows low background noise, but due to the optical pathlength‐dependence of the photothermal signal, sensitivity of DIC‐PTM in an ultrathin channel is still low. To enhance the sensitivity, organic solvents have been used according to the photothermal signal enhancement factor (Shimizu et al ., ), and/or heat isolation layer has been added into the chip to confine the heat in extended nanofluidic channels (Le et al ., ). A limit of detecion (LOD) of 2.4 M of Sunset Yellow dye, corresponding to 390 molecules in a detection volume of 0.25 fL, was achieved in a 21 m wide ×500 nm deep nanochannel (Shimizu et al ., ).…”