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We have developed a next-generation, non-fluorescent, single-molecule technique by integrating a single biomolecule with a carbon-nanotube field effect transistor. This approach has opened up an entirely new way of watching, recording, and analyzing single biochemical events happening in real time. By using this new tool’s outstanding sensitivity and temporal resolution, our research aims to probe the unknown molecular mechanisms and functions of enzymes involved human diseases including cancer.
We have developed a next-generation, non-fluorescent, single-molecule technique by integrating a single biomolecule with a carbon-nanotube field effect transistor. This approach has opened up an entirely new way of watching, recording, and analyzing single biochemical events happening in real time. By using this new tool’s outstanding sensitivity and temporal resolution, our research aims to probe the unknown molecular mechanisms and functions of enzymes involved human diseases including cancer.
Single molecule nanocircuits: (A) Schematic diagram of the single lysozyme being interrogated by a carbon nanocircuit. The partial poly(methyl methacrylate) coating is depicted in gray. (B) AFM topography of a SWNT FET before (inset) and after coating with the pyrene linker, lysozyme incubation, and washing to reduce nonspecific binding. The circle highlights the point of lysozyme attachment. (C) Response of current in a lysozyme device to electrolytic gating. (D) I(t) measured in phosphate buffer, with peptidoglycan substrate (25 mg/ml) added to the solution at t = 0. The inset with a magnified time axis indicates a rapid response of <50 ms (inset).
Single molecule nanocircuits: (A) Schematic diagram of the single lysozyme being interrogated by a carbon nanocircuit. The partial poly(methyl methacrylate) coating is depicted in gray. (B) AFM topography of a SWNT FET before (inset) and after coating with the pyrene linker, lysozyme incubation, and washing to reduce nonspecific binding. The circle highlights the point of lysozyme attachment. (C) Response of current in a lysozyme device to electrolytic gating. (D) I(t) measured in phosphate buffer, with peptidoglycan substrate (25 mg/ml) added to the solution at t = 0. The inset with a magnified time axis indicates a rapid response of <50 ms (inset).
Single-molecule enzyme studies. Active-site conformational motions are essential for the catalytic function of T4 lysozyme . Through open-close motions, the enzyme adjusts its physical and chemical flexibility toward an active state, which consists of the binding complex of an enzyme and a substrate for a specific catalytic reaction. The SWNT electronic measurements of single-molecule T4 lysozyme conformational dynamics (B) allow longer trajectories to be recorded than with FRET spectroscopic studies (C).
Single-molecule enzyme studies. Active-site conformational motions are essential for the catalytic function of T4 lysozyme . Through open-close motions, the enzyme adjusts its physical and chemical flexibility toward an active state, which consists of the binding complex of an enzyme and a substrate for a specific catalytic reaction. The SWNT electronic measurements of single-molecule T4 lysozyme conformational dynamics (B) allow longer trajectories to be recorded than with FRET spectroscopic studies (C).
Electronic monitoring of protein-substrate interaction. (A) Long-duration I(t) sequences exhibit dynamic noise on top of low-frequency fluctuations (yellow line) having a 1/f distribution. (B) Subtracting the meandering mean produces a filtered data set that clarifies the fluctuations as two-level, simplifies further analysis, and reveals that the two-level switching rates vary over 5- to 15-s periods. (C) The faster RTS oscillates about 300 times per second, whereas (D) the slower RTS oscillates 15 times per second. The insets show individual switching events for each case.
Electronic monitoring of protein-substrate interaction. (A) Long-duration I(t) sequences exhibit dynamic noise on top of low-frequency fluctuations (yellow line) having a 1/f distribution. (B) Subtracting the meandering mean produces a filtered data set that clarifies the fluctuations as two-level, simplifies further analysis, and reveals that the two-level switching rates vary over 5- to 15-s periods. (C) The faster RTS oscillates about 300 times per second, whereas (D) the slower RTS oscillates 15 times per second. The insets show individual switching events for each case.
Currently, we are probing dynamic features of protein-protein and protein-ligand interactions during enzyme catalysis.
Currently, we are probing dynamic features of protein-protein and protein-ligand interactions during enzyme catalysis.
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