Optofluidic restricted imaging, spectroscopy and counting of nanoparticles by evanescent wave using immiscible liquids.

Conventional flow cytometry (FC) suffers from the diffraction limit for the detection of nanoparticles smaller than 100 nm, whereas traditional total internal reflection (TIR) microscopy can only detect few samples near the solid-liquid interface mostly in static states. Here we demonstrate a novel on-chip optofluidic technique using evanescent wave sensing for single nanoparticle real time detection by combining hydrodynamic focusing and TIR using immiscible flows. The immiscibility of the high-index sheath flow and the low-index core flow naturally generate a smooth, flat and step-index interface that is ideal for the TIR effect, whose evanescent field can penetrate the full width of the core flow. Hydrodynamic focusing can focus on all the nanoparticles in the extreme centre of the core flow with a width smaller than 1 μm. This technique enables us to illuminate every single sample in the running core flow by the evanescent field, leaving none unaffected. Moreover, it works well for samples much smaller than the diffraction limit. We have successfully demonstrated the scattering imaging and counting of 50 nm and 100 nm Au nanoparticles and also the fluorescence imaging and counting of 200 nm beads. The effective counting speeds are estimated as 1500, 2300 and 2000 particles per second for the three types of nanoparticles, respectively. The optical scattering spectra were also measured to determine the size of individual Au nanoparticles. This provides a new technique to detect nanoparticles and we foresee its application in the detection of molecules for biomedical analyses.