Ultra-sensitive Refractometry via Supercritical Angle Fluorescence Analysis

The refractive index (RI) is a material property that describes its interaction with light. This can be used to extract information about concentrations, material structure, and much more. Recently, sensitive methods for RI measurements of small volumes have been applied to detection of pathogens and quantification of protein-ligand interactions. Here we describe a method for label-free sensing of the RI: a device composed of a glass coverslip coated with a thin, dense layer of fluorophores inside a microfluidics channel. The device is imaged using a fluorescence microscope, where the detector is placed in a conjugate back focal plane of a high-NA objective lens. The RI is then calculated using a robust, custom algorithm for determining the transition between the undercritical and supercritical fluorescence. This is done by an iterative circle fitting method that estimates the radius of maximum intensity in the observed pattern, which scales linearly with the RI of the medium.

We validate the methodology over a broad range of indices with ultra-sensitivity. Our microfluidic-based device and analysis technique for refractometry, enable label-free measurements of tiny amounts of liquid sample (picolitres of imaged volume) with high throughput and an order of magnitude higher precision than was previously reported. The sensitivity of our device enables detection of RI changes smaller than 4e-5 RI units, corresponding to a change of less than a 0.1% glycerol concentration in a water-glycerol solution. We furthermore demonstrate our system’s applicability for biosensing, by measuring E. coli bacteria growth in the chamber.