Frequency locked whispering evanescent resonator (FLOWER) for biochemical sensing applications.

Sensitive, rapid and label-free biochemical sensors are needed for many applications. In this protocol, we describe biochemical detection using FLOWER (frequency locked optical whispering evanescent resonator)-a technique that we have used to detect single protein molecules in aqueous solution as well as exosomes, ribosomes and low part-per-trillion concentrations of volatile organic compounds. Whispering gallery mode microtoroid resonators confine light for extended time periods (hundreds of nanoseconds). When light circulates within the resonator, a portion of the electromagnetic field extends beyond the cavity, forming an evanescent field. This field interacts with bound analytes resulting in a change in the cavity's effective refractive index, which can be tracked by monitoring shifts in the resonance wavelength. The surface of the microtoroid can be functionalized to respond specifically to an analyte or biochemical interaction of interest. The frequency-locking feature of frequency locked optical whispering evanescent resonator means that the instruments respond to perturbations in the surface by very rapidly finding the new resonant frequency. Here we describe microtoroid fabrication (4-6 h), how to couple light into these devices using tapered optical fibers (20-40 min) and procedures for coupling antibodies as well as G-protein coupled receptors to the microtoroid's surface (from 1 h to 1 d depending on the target analyte). In addition, we describe our liquid handling perfusion system as well as the use of a rotary selector valve and custom fluidic chamber to optimize sample delivery. Step-by-step details on how to perform biosensing experiments and analyze the data are described; this takes 1-2 d.