Measuring FRAP in engineered cell membranes
EMSL Project ID
60585
Abstract
We propose Threat Agnostic Sensing Using Biomimetic Devices. There is a clear need for simple, fast, and robust toxin activity and pathogen detection assays without use of live cells, that can cover a range of targets and provide specific information on the mode of action (e.g. binding, blocking, or disruption) present in a sample. Robust methods for expression and manipulation of functional toxin receptors are needed to enable advancement of capabilities for field deployable detection assays and development of countermeasures. In contrast to bacterial pore proteins that have been widely used in measurement devices, the larger and more complex mammalian membrane proteins that toxins act on have generally not been used in this way. Instead, they have been studied using the “patch clamp” approach, which is expensive, time intensive, and can lack reproducibility between technicians. Previous efforts through the Friend or Foe Program sponsored by DARPA have led to the development of biomimetic organic electronic devices for characterizing host-pathogen interactions. This technology provides an authentic membrane and sensing strategy that is robust, sensitive, captures the necessary complexity, and is scalable. These membranes, referred to as supported lipid bilayers (SLBs) are harvested from cells expressing the receptor of interest, then deposited onto an electronic device that can provide impedance and transient electrical responses of membranes. This cell free approach has the potential to faithfully recapitulate the response of a live cell, and the bioelectronic devices have been shown to have a shelf life of approximately 3 weeks. More recent work has been focused on ion channels that are expressed at the level of the membrane, which are important toxin targets.
Fluorescence recovery after photobleaching (FRAP) is a method to study the mobility of molecules in the cell membrane. We propose to engineer human embryonic kidney (HEK) cell lines with ion channels and receptors that are expressed at the cellular membrane. The membrane of these cells will then be harvested and deposited onto a biomimetic device to test their ability to respond to toxic insults. To ensure these membranes are functional after harvesting, we will conduct FRAP assays to ensure molecules will be mobile within the membrane itself. Utilization of EMSL resources, specifically the Airyscan module that is mounted on the confocal microscope, will enable visualization of fluorescent molecules moving through the membrane. The data captured from this resource will provide generation of high-quality data with respect to the integrity of harvested membranes that are used in downstream applications.
Project Details
Start Date
2022-10-17
End Date
2023-09-30
Status
Closed
Released Data Link
Team
Principal Investigator