Non-invasive Electrophysiology of Individual Microbes in a Colony in Real-time
Abstract
Coupling the machinery of a microorganism to physical electronics is a profoundly attractive avenue for making electronic circuitry responsive to complex environmental change. A tangible avenue is gating a transistor by microbeÕs cell membrane potential. This is an ideal electronic device element to tap the complex biochemistry of the cell to drive an electronic device without any electron exchange. However, driving a transistor (i.e., gating) with microorganisms has not been possible due to their thick, nonconformal cell wall. To our knowledge, we have made the first living electrochemical field-effect transistor (LeFET) coupled to the photosynthesis of microalgae and single virus infection that demonstrates single-cell sensitivity. The LeFET quantitatively measures the modulation of cell membrane potential in real time. The devices were individually fabricated by local dispensing of adhesive to hermetically seal them for operating in liquid. The goal of the proposed program is to take the next step in microfabricating a living hybrid chip with a network of LeFET devices that interfaces with a colony of microbes where each device is gated by a single cell in the population. The key innovation is the special architecture of the device channelÑthe nanoparticle necklace network (N3). N3 is made in a twostep process: One-dimensional (1D) necklaces of 10 nm gold (Au) particles are made by directed self-assembly. The necklaces are deposited by centrifuge to form a self-limited monolayer to form a 2D network. In the ARO/STIR program we demonstrated a unique key feature of N3 gating. Unlike nanoparticle array devices, the architecture exhibits a constant conduction gap on gating. The constant gap led to universal gating behavior which to our knowledge has not been observed before in percolating systems. As a result, in the ARO/STIR program we demonstrated a gain of 103 compared to less than 10 for metallic nanoparticle array based transistors. In the proposed study the high gain will be leveraged to develop a MicroBio Chip to address biochemical activity of a microbe colony at single cell level. The study will focus on the highly versatile and pervasive phenomenon of persister state by which bacteria gain antibiotic resistance. Persister state dormancy is observed in all bacteria where antibiotic efflux activity, such as a resistance-nodulation-division (RND) pump is enhanced to evade toxins. It is a phenotypic change that is not genetic or heritable, so the cell can be reversibly switched back to a non-persister state to subsequently propagate infection. Because an RND is driven by proton motive force, its operation will lead to large modulation in cell membrane potential to cause significant gating. The proposed research will be to choose antibiotics with macroscopic fluorescence signal to benchmark and validate the LeFET performance. Formation of persister state, its resurrection, and cell-cell communication during antibiotic stress will be studied in real-time. Specifically, MicroBio Chip will study the cellular response from individual Escherichia coli cells in a heterogeneous population in persister and non-persister states as they combats antibiotics and resurrect on the removal of the toxin. The overarching goal will be to develop a highly sensitive, quantitative technology to study electrophysiology of microbes and their complex behavior, such as, development of antibiotic resistance. The MicroBio Chip will have application in rapidly screening for patient specific antibiotics for sepsis therapy. Furthermore, the MicroBio Chip with engineered microbes could be developed to make complex adaptable hybrid systems with evolutionary response as the cells adapt. The evolutionary response could be coupled to machine learning algorithms to develop intelligent sensing algorithms. The chip would be a tool for fundamental electro-microbiology research on a cell population (not possible today).
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Feb 01, 2023
- Source ID
- W911NF2310036
Entities
People
- Ravi F Saraf
Organizations
- Army Contracting Command
- United States Army
- University of Nebraska–Lincoln