Biosensing within complex biological samples requires a sensor that can compensate for fluctuations in the signal due to changing environmental conditions and nonspecific binding events. To achieve this, we developed a novel self-referenced biosensor consisting of two almost identically sized dye-doped polystyrene microspheres placed on adjacent holes at the tip of a microstructured optical fiber (MOF). Here self-referenced biosensing is demonstrated with the detection of Neutravidin in undiluted, immunoglobulin-deprived human serum samples. The MOF allows remote excitation and collection of the whispering gallery modes (WGMs) of the microspheres while also providing a robust and easy to manipulate dip-sensing platform. By taking advantage of surface functionalization techniques, one microsphere acts as a dynamic reference, compensating for nonspecific binding events and changes in the environment (such as refractive index and temperature), while the other microsphere is functionalized to detect a specific interaction. The almost identical size allows the two spheres to have virtually identical refractive index sensitivity and surface area, while still having discernible WGM spectra. This ensures their responses to nonspecific binding and environmental changes are almost identical, whereby any specific changes, such as binding events, can be monitored via the relative movement between the two sets of WGM peaks.
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