Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process.

Serotonin is an important neurotransmitter involved in various functions of the nervous, blood, and immune system. In general, detection of small biomolecules such as serotonin in real time with high spatial and temporal resolution remains challenging with conventional sensors and methods. In this work, we designed a near-infrared (nIR) fluorescent nanosensor (NIRSer) based on fluorescent singlewalled carbon nanotubes (SWCNTs) to image the release of serotonin from human blood platelets in real time. The nanosensor consists of a nonbleaching SWCNT backbone, which is fluorescent in the beneficial nIR tissue transparency window (800−1700 nm) and a serotonin binding DNA aptamer. The fluorescence of the NIRSer sensor (995 nm emission wavelength for (6,5)-SWCNTs) increases in response to serotonin by a factor up to 1.8. It detects serotonin reversibly with a dissociation constant of 301 nM ± 138 nM and a dynamic linear range in the physiologically relevant region from 100 nM to 1 μM. As a proof of principle, we detected serotonin release patterns from activated platelets on the single-cell level. Imaging of the nanosensors around and under the platelets enabled us to locate hot spots of serotonin release and quantify the time delay (≈ 21−30 s) between stimulation and release in a population of platelets, highlighting the spatiotemporal resolution of this nanosensor approach. In summary, we report a nIR fluorescent nanosensor for the neurotransmitter serotonin and show its potential for imaging of chemical communication between cells.

Detection of neurotransmitters is an analytical challenge and essential to understand neuronal networks in the brain and associated diseases. However, most methods do not provide sufficient spatial, temporal, or chemical resolution. Near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) have been used as building blocks for sensors/probes that detect catecholamine neurotransmitters, including dopamine. This approach provides a high spatial and temporal resolution, but it is not understood if these sensors are able to distinguish dopamine from similar catecholamine neurotransmitters, such as epinephrine or norepinephrine. In this work, the organic phase (DNA sequence) around SWCNTs was varied to create sensors with different selectivity and sensitivity for catecholamine neurotransmitters. Most DNA-functionalized SWCNTs responded to catecholamine neurotransmitters, but both dissociation constants (Kd) and limits of detection were highly dependent on functionalization (sequence). Kd values span a range of 2.3 nM (SWCNT-(GC)15 + norepinephrine) to 9.4 μM (SWCNT-(AT)15 + dopamine) and limits of detection are mostly in the single-digit nM regime. Additionally, sensors of different SWCNT chirality show different fluorescence increases. Moreover, certain sensors (e.g., SWCNT-(GT)10) distinguish between different catecholamines, such as dopamine and norepinephrine at low concentrations (50 nM). These results show that SWCNTs functionalized with certain DNA sequences are able to discriminate between catecholamine neurotransmitters or to detect them in the presence of interfering substances of similar structure. Such sensors will be useful to measure and study neurotransmitter signaling in complex biological settings.

Single-walled carbon nanotubes (SWCNTs) have unique photophysical properties and promise many novel applications. Their functionalization is crucial, but the organic phase around SWCNTs is poorly understood. Noncovalent functionalization with single-stranded DNA (ssDNA) is one of the most used approaches to solubilize SWCNTs in water, and variation of ssDNA sequences leads to major advances in separation of SWCNT chiralities and SWCNT-based sensors. However, the exact number of adsorbed ssDNA molecules on ssDNA/SWCNT complexes and consequently the surface coverage are not precisely known. Here, we determine the number of adsorbed/bound ssDNA molecules per SWCNT for different ssDNA sequences. For this purpose, we directly quantify free and bound/adsorbed ssDNA and the concentration of SWCNTs using an approach based on filtration, absorption spectroscopy, and atomic force microscopy. We found that the number of adsorbed ssDNA molecules on 600 nm long (6,5)-SWCNTs varies between ∼860 for (GT)5 and ∼130 for (A)30. Interestingly, there are large differences in the average SWCNT segment lengths one ssDNA molecule occupies. It varies between sequences of the same length from ∼2.1 nm (T)30 and ∼3.2 nm (C)30 to ∼4.6 nm (A)30. The sequence (GT)15 occupied an average SWCNT segment of ∼2.3 nm. In contrast, for (GT) x repeats, we found a linear decrease of the number of adsorbed ssDNA molecules with sequence length x. We also correlated surface occupation with the ssDNA/SWCNT near-infrared (nIR) fluorescence responses to analytes such as dopamine, H2O2, riboflavin and pH changes. In most cases, the nIR fluorescence responses did not correlate with the number of adsorbed ssDNA molecules, indicating that the exact structure of the crowded corona around the SWCNT is crucial for photophysical changes. The occupied SWCNT segment length per ssDNA molecule is also an important parameter for molecular dynamics (MD) simulations. (GT)15 ssDNA adsorbed and stacked on top of each, when using the determined parameters, in contrast to simulations with an excess SWCNT surface. This result highlights the importance of knowing the number of adsorbed ssDNA molecules per SWCNT. In summary, we present a versatile and direct assay to determine the amount of ssDNA molecules adsorbed on SWCNTs, report those numbers, and evaluate how they are related to photophysical properties.