Label-free, real-time monitoring of membrane binding events at zeptomolar concentrations using frequency-locked optical microresonators

Abstract

Binding events to elements of the cell membrane act as receptors which regulate cellular function and communication and are the targets of many small molecule drug discovery efforts for agonists and antagonists. Conventional techniques to probe these interactions generally require labels and large amounts of receptor to achieve satisfactory sensitivity. Whispering gallery mode microtoroid optical resonators have demonstrated sensitivity to detect single-molecule binding events. Here, we demonstrate the use of frequency-locked optical microtoroids for characterization of membrane interactions in vitro at zeptomolar concentrations using a supported biomimetic membrane. Arrays of microtoroids were produced using photolithography and subsequently modified with a biomimetic membrane, providing high quality (Q) factors ($>{10}^6$) in aqueous environments. Fluorescent recovery after photobleaching (FRAP) experiments confirmed the retained fluidity of the microtoroid supported-lipid membrane with a diffusion coefficient of $3.38\pm 0.26{\text{μm}}^2\cdot {\text{s}}^{-1}$. Utilizing this frequency-locked membrane-on-a-chip model combined with auto-balanced detection and non-linear post-processing techniques, we demonstrate zeptomolar detection levels The binding of Cholera Toxin B- monosialotetrahexosyl ganglioside (GM1) was monitored in real-time, with an apparent equilibrium dissociation constant $\left(k_{\text{d}}\right)=1.53\text{nM}$. The measured affiny of the agonist dynorphin A 1-13 to the $\text{κ}$-opioid receptor revealed a $k_{\text{d}}=3.1\text{nM}$ using the same approach. Radioligand binding competition with dynorphin A 1-13 revealed a $k_{\text{d}}$ in agreement (1.1 nM) with the unlabeled method. The biosensing platform reported herein provides a highly sensitive real-time characterization of membrane embedded protein binding kinetics, that is rapid and label-free, for toxin screening and drug discovery, among other applications.