Activity

After cleaning, the membrane properties were unaffected; this, combined with numerous pressurizing cycles, demonstrated reusability of the system.Recent advances in fluorescence microscopy allow us to track chemical reactions at the single-molecule level. Single-molecule measurements make it possible to minimize the amount of sample needed for analysis and diagnosis. Signal amplification is often applied to ultralow-level biomarker detection. Polymerase chain reaction (PCR) is used to detect DNA/RNA, and enzyme-linked immunosorbent assay (ELISA) can sensitively probe antigen-antibody interactions. While these techniques are brilliant and will continue to be used in the future, single-molecule-level measurements would allow us to reduce the time and cost needed to amplify signals.The kinetics of chemical reactions have been studied mainly using ensemble-averaged methods. However, they can hardly distinguish time-dependent fluctuations and static heterogeneity of the kinetics. The information hidden in ensemble-averaged measurements would be extractable from a single-molecule experiment. Thus, single-molecule measurement would provide unique opportunitie and resolved from the random background signal.In this Account, we summarize the recent studies on the single-molecule measurement of redox reaction kinetics, with a focus on our group’s recent progress. We first introduce the control of redox blinking to increase the photostability of fluorescent molecules. We then demonstrate the control of redox blinking, which allows us to detect target DNA by monitoring the function of a molecular beacon-type probe, and we investigate antigen-antibody interactions at the single-molecule level. By tracing the time-dependent changes in blinking patterns, redox blinking is shown to be adaptable to tracking the structural switching dynamics of RNA, the preQ1 riboswitch. This Account ends with a discussion of our ongoing work on the control of fluorescent blinking. We also discuss the development of devices that allow single-molecule-level analysis in a high-throughput fashion.Flexible pressure sensors have attracted increasing attention because they can mimic human skin to sense external pressure; however, for mimicking human skin, the sensing of a pressure point is far from sufficient. To realize fully biomimetic skins, it is crucial for flexible sensors to have high resolution and high sensitivity. We conducted simulations and experiments to determine the relationship between the sensor sensitivity and physical parameters, such as the effective relative permittivity and air ratio of the dielectric layer. According to the results, a micropillar-poly(vinylidene fluoride) (PVDF) dielectric layer was designed to achieve high sensitivity (0.43 kPa-1) in the low-pressure regime ( less then 1 kPa). An 8 × 8 pixel sensor matrix was prepared based on a micropillar-PVDF (MP) film and electrode array (MPEA) to detect the pressure distribution with high resolution (13 dpi). Each pixel could reflect the point of applied pressure through an obvious change in the relative capacitance; moreover, objects with various geometries could be mapped by the pixels of the flexible sensor. A counterweight, a plastic flag, and pine leaves were placed on the flexible sensor, and the shapes were successfully mapped; in particular, the mapping of the ∼0.005 g ultra-lightweight pine leaves with a length of 7 mm and a width of 0.6 mm shows the high sensitivity and high resolution of our flexible pressure sensor.Macrocyclic peptides open new opportunities to target intracellular protein-protein interactions (PPIs) that are often considered nondruggable by traditional small molecules. However, engineering sufficient membrane permeability into these molecules is a central challenge for identifying clinical candidates. Currently, there is a lack of high-throughput assays to assess peptide permeability, which limits our capacity to engineer this property into macrocyclic peptides for advancement through drug discovery pipelines. 5-Ethynyluridine supplier Accordingly, we developed a high throughput and target-agnostic cell permeability assay that measures the relative cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay was named NanoClick as it combines in-cell Click chemistry with an intracellular NanoBRET signal. We validated the approach using known cell penetrating peptides and further demonstrated a correlation to cellular activity using a p53/MDM2 model system. With minimal change to the peptide sequence, NanoClick enables the ability to measure uptake of molecules that enter the cell via different mechanisms such as endocytosis, membrane translocation, or passive permeability. Overall, the NanoClick assay can serve as a screening tool to uncover predictive design rules to guide structure-activity-permeability relationships in the optimization of functionally active molecules.Demyelination, the loss of the protecting sheath of neurons, contributes to disability in many neurological diseases. In order to fully understand its role in different diseases and to monitor treatments aiming at reversing this process, it would be valuable to have PET radiotracers that can detect and quantify molecular changes involved in demyelination such as the uncovering and upregulation of the axonal potassium channels Kv1.1 and Kv1.2. Carbon-11 labeled radiotracers present the advantage of allowing for multiple scans on the same subject in the same day. Here, we describe [11C]3MeO4AP, a novel 11C-labeled version of the K+ channel tracer [18F]3F4AP, and characterize its imaging properties in two non-human primates including a monkey with a focal brain injury sustained during a surgical procedure 3 years prior to imaging. Our findings show that [11C]3MeO4AP is brain permeable, metabolically stable and has high plasma availability. When compared with [18F]3F4AP, [11C]3MeO4AP shows very high correlation in volumes of distribution (VT), confirming a common target. [11C]3MeO4AP shows slower washout than [18F]3F4AP, suggesting stronger binding. Finally, similar to [18F]3F4AP, [11C]3MeO4AP is highly sensitive to the focal brain injury. All these features make it a promising radioligand for imaging demyelinated lesions.