Distinguishing Signal Contributions from Bulk Fluid and Channel Surface Regions to Improve Backscattering Interferometry

Abstract

Backscattering Interferometry (BSI) has been used to detect bio-molecular interactions by measuring shifts in the bulk refractive index of a sample due to conformational changes of the interacting species. BSI has the advantage over other biosensors because it can detect interactions in a free solution format, as well as a tethered format. In some applications, non-specific surface adsorption introduces noise to the BSI signal, negatively affecting its limit of detection. We hypothesize that there are regions within the spatial and frequency domain of the BSI signal, which are only sensitive to changes in the bulk fluid, and are thus unaffected by interactions on the surface of the channel. To test this hypothesis, model systems were designed in which the surface conditions of a glass microchannel, as well as the bulk fluid, were changed in a controlled manner. Step changes in glycerol concentration in the bulk fluid were made and successive chemical layers were coupled to the surface to produce independent bulk and surface signals. Regions of the fringe pattern produced by BSI, which have been previously uninvestigated, were analyzed to determine if there are distinct characteristics of the signal that change in response to bulk refractive index but are insensitive to surface binding. We developed an algorithm to identify these regions, effectively eliminating the unwanted signal contributions from surface adhesion while maintaining the signal produced by the bulk fluid. Multiple regions, within the parameter space of each model, were found that are insensitive to surface binding molecules.