Improving Stability and Preparation Efficiency of Electrophysiological Sensors

Abstract

Pipet aperture-based electrophysiological (EP) sensors have emerged relatively recently as powerful bioanalytical tools. Taking advantage of the ligand specificity and inherent high sensitivity of membrane proteins, pipet aperture-based EP sensors can transduce target analyte binding events into measurable electrical signals. A variety of analytes including neurotransmitters and hormones that traditional fluorescence or electrochemistry methods are ineffective for detection can be quantitatively measured, facilitated by biologically “paired” receptor membrane proteins. Additionally, such sensors intrinsically provide high spatial resolution due to the pipet apertures that can be fabricated on µm scales, enabling real-time cellular secretion measurements. Despite these strengths, applications that aim to utilize such sensors have been limited, predominately due to low efficiency in sensor preparation. First, the preparation of some sensor components, such as membrane proteins, is typically laborious and time-consuming, resulting in a low success rate and a long timescale of sensor assembly and functionalization. Second, artificial lipid membrane platforms for membrane proteins have low stability, leading to early rupture and short sensor longevity. This requires reproduction of sensors of the same type to achieve measurements that could have been performed with fewer but more stable individual sensors. Therefore, improving sensor preparation efficiency could significantly broaden applications of EP sensors. This dissertation investigates these two aspects of sensor preparation efficiency and demonstrates two ways to apply such sensors in measuring small molecules and studying peptides. To stabilize the membrane, methacrylate polymerization with various initiation methods was adopted (Chapter 2). To increase membrane protein preparation efficiency, in vitro transcription and translation was utilized to overcome limitations in traditional protein expression and purification workflows (Chapter 4). Regarding applications, Chapter 3 demonstrates the preparation, calibration, and evaluation of pipet aperture-based EP sensors with tunable extent of membrane stabilization. Chapter 5 elucidates how such sensors can be used to characterize pore-forming peptides, mainly regarding the functional stoichiometries. Overall, improvement, development, and applications of pipet aperture-based EP sensors were explored.