Insulated conducting cantilevered nanotips and two-chamber recording system for high resolution ion sensing AFM.

Biological membranes contain ion channels, which are nanoscale pores allowing controlled ionic transport and mediating key biological functions underlying normal/abnormal living. Synthetic membranes with defined pores are being developed to control various processes, including filtration of pollutants, charge transport for energy storage, and separation of fluids and molecules. Although ionic transport (currents) can be measured with single channel resolution, imaging their structure and ionic currents simultaneously is difficult. Atomic force microscopy enables high resolution imaging of nanoscale structures and can be modified to measure ionic currents simultaneously. Moreover, the ionic currents can also be used to image structures. A simple method for fabricating conducting AFM cantilevers to image pore structures at high resolution is reported. Tungsten microwires with nanoscale tips are insulated except at the apex. This allows simultaneous imaging via cantilever deflections in normal AFM force feedback mode as well as measuring localized ionic currents. These novel probes measure ionic currents as small as picoampere while providing nanoscale spatial resolution surface topography and is suitable for measuring ionic currents and conductance of biological ion channels

Light-Controlled Cell–Cell Assembly Using Photocaged Oligonucleotides

Article asserts that while techniques that allow one to control the arrangement of cells and direct contact between different cell types have been developed that expand upon simple co-culture methods, specific control over heterojunctions that form between cells is not easily accomplished with current methods, such as 3D cell-printing. In this article, DNA-mediated cell interactions are combined with cell-compatible photolithographic approaches to control cell assembly

Design and Development of Integrated Multi-Modal Scanning Probe Micrscopy for Structure-Function Imaging of Ion Channels and Receptors

Understanding cellular behavior and tissue organization requires a deeper understanding of nanoscale structure and activity of proteins and biomacromolecules, including channels and receptors that act as messengers connecting the cell to its surrounding. Channels and receptors respond to a wide variety of electrical, chemical, and mechanical stimuli to facilitate cellular homeostasis, communication, migration, and survival. Ion channels facilitate the passage of ions and metabolites across cellular membranes and are visualized by high-resolution 3D imaging techniques, which include EM and scanning probe microscopies, such as atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM). However, the current imaging techniques are unable to obtain the intertwined direct relationships between structure and electrical activity of ion channels. My work is dedicated to designing and implementing such an integrated system. This dissertation describes the details about different novel AFM-based nanotechnologies designed and developed for simultaneous structure-activity imaging of various electrically active biological systems. First, a novel AFM probe was developed by insulating tungsten micro-wires, which can measure electrical activity at the nanoscale. These probes, coated in gold, were used to image the structure of Escherichia coli that surface express mutants of the redox active enzyme, alcohol dehydrogenase II. Simultaneous structure-function imaging of the bacteria cells revealed improved electron transfer when mediators were placed closer to the NADH binding pocket. Second, a two-chamber system mimicking biological membranes (~5 nm thick) that enables the imaging of ion channel proteins in lipid membrane models was developed. The two chambers were separated by a 5 nm thick insulated graphene sheet deposited over a 1 μm hole. A TEM was used to drill a ~20 nm pore. The substrate supports lipid membranes for measuring electrical activity. Third, AFM was used to image cell-surround communication channels (Connexin26 hemichannels) in purified membrane plaques as well as in reconstituted lipid membranes revealing channel clustering in high-resolution images. The electrical activity of these hemichannel preparations were then recorded when de- posited over the nanopore supports for initial simultaneous electrical recording and imaging. Lastly, the design and development of a parallel SICM-array capable of simultaneous multi-point high-throughput nanoscale imaging was realized

Measuring localized redox enzyme electron transfer in a live cell with conducting atomic force microscopy.

Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of E. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells