Methods for High-Contrast Photoluminescence and Label-Free Surface-Sensitive Detection

Abstract

Physical phenomena such as total internal reflection, scattering and interference, or plasmon resonances enable surface-sensitive detection methods. These methods have high sensitivities that allow the detection of single nanoparticles or single molecules such as proteins. Proteins have interesting physiological functions. One example are kinesins that are a key to essential biological processes such as cell division and cellular transport. Kinesins are motor proteins that move along microtubule “tracks”. To study single kinesins, they are conventionally labeled with fluorescent dyes and detected using total internal reflection fluorescence (TIRF) microscopy. However, to improve the understanding of their native interaction with microtubules, a label-free detection is desirable. Due to the large size of the kinesin molecule, label-free detection can be envisioned by optimized interference reflection or interferometric scattering microscopy (IRM and iSCAT). Alternatively, unlabeled kinesins could potentially be detected using the highly sensitive near-field of plasmonic nanoparticles. One limiting factor of all surface-sensitive detection techniques is the requirement of a high quality surface. Especially for biological samples, the assays become complex while exhibiting a short storage life. Therefore, well functionalized surfaces must be prepared frequently. Here, I developed a method to generate reproducibly high quality surfaces for high contrast imaging of microtubules and kinesins with a minimized work effort. With the new protocol, molecularly clean surfaces are obtained that are reliably functionalized and yield low-background microfluidic chambers suited for all further measurements. To enable label-free detection of kinesins in the future, I wrote an image acquisition and analysis program that allows real-time iSCAT imaging with simultaneous IRM detection. Additionally, I developed a method that enabled the binding of gold nanorods to microtubules, without blocking their highly sensitive plasmonic near-field. Measurements suggested that these functionalized plasmonic nanoantennas could serve as multipurpose bleach and blink-free in vitro microtubule markers. In the future, with improved surface preparations and label-free techniques, microtubule-bound plasmonic antennas potentially can be used as ultrasensitive single-molecule sensors for molecular machines translocating along microtubules