The near-infrared (nIR) fluorescence of polymer-wrapped single-walled carbon nanotubes (SWCNTs) is very sensitive to the local chemical environment. It has been shown that certain small reducing molecules can increase the fluorescence of SWCNTs. However, so far the role of the polymer around the SWCNT as well as the mechanism is not understood. Here, we investigated how reducing and oxidizing small molecules affect the nIR fluorescence of polymer-wrapped SWCNTs. Our results show that the polymer plays an essential role. Reducing molecules such as ascorbic acid, epinephrine, and trolox increased the nIR fluorescence up to 250% but only if SWCNTs were suspended in negatively charged polymers such as DNA or poly(acrylic acid) (PAA). In comparison, phospholipid−poly(ethylene glycol) wrapped SWCNTs did not respond at all while positively charged polyallylamine-wrapped SWCNTs were quenched. Oxidized equivalents such as dehydroascorbic acid did not show a clear tendency to quench or increase fluorescence. Only riboflavin with an intermediate oxidation potential and light absorption in the visible range quenched all polymerwrapped SWCNTs. In general, polymer-wrapped SWCNTs that responded to reducing molecules (e.g., +141%, ascorbic acid) also responded to oxidizing molecules (e.g., −81%, riboflavin). Nevertheless, several reducing molecules showed only a small fluorescence increase (NADH, +21%) or even a decrease (glutathione, −14%), which highlights that the redox potential alone cannot explain fluorescence changes. Furthermore, we show that neither changes of absorption cross sections, scavenging of reactive oxygen species (ROS), nor free surface areas on SWCNTs explain the observed patterns. However, results are in agreement either with a redox reaction of the polymer or conformational changes of the polymer that change fluorescence decay routes. In summary, we show that the polymer around SWCNTs governs how redox-active molecules change nIR fluorescence (quantum yield) of SWCNTs. Molecules with a low redox potential (<−0.4 V) are more likely to increase SWCNT fluorescence, but a low redox-potential alone is not sufficient.
The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5–10%), heat-inactivated (hiFCS, 0.1–10%) or human serum albumin (HSA, 0.05–2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.
Integrins are transmembrane receptors that mediate cell-adhesion, signaling cascades and platelet-mediated blood clotting. Most integrins bind to the common short peptide Arg-Gly-Asp (RGD). The conformational freedom of the RGD motif determines how strong and to which integrins it binds. Here, we present a novel approach to tune binding constants by confining RGD peptide motifs via noncovalent adsorption of single-stranded DNA (ssDNA) anchors onto single-walled carbon nanotubes (SWCNTs). Semiconducting SWCNTs display fluorescence in the near-infrared (nIR) region and are versatile fluorescent building blocks for imaging and biosensing. The basic idea of this approach is that the DNA adsorbed on the SWCNT surface determines the conformational freedom of the RGD motif and affects binding affinities. The RGD motif was conjugated to different ssDNA sequences in both linear ssDNA-RGD and bridged ssDNA-RGD-ssDNA geometries. Molecular dynamics (MD) simulations show that the RGD motif in all the synthesized systems is mostly exposed to solvent and thus available for binding, but its flexibility depends on the exact geometry. The affinity for the human platelet integrin αβ could be modulated up to 15-fold by changing the ssDNA sequence. IC values varied from 309 nM for (C)-RGD/SWCNT hybrids to 29 nM for (GT)-RGD/SWCNT hybrids. When immobilized onto surface adhesion of epithelial cells increased 6-fold for (GT)-RGD/SWCNTs. (GT)-RGD/SWCNTs also increased the number of adhering human platelets by a factor of 4.8. Additionally, αβ integrins on human platelets were labeled in the nIR by incubating them with these ssDNA-peptide/SWCNT hybrids. In summary, we show that ssDNA-peptide hybrid structures noncovalently adsorb onto SWCNTs and serve as recognition units for cell surface receptors such as integrins. The DNA sequence affects the overall RGD affinity, which is a versatile and straightforward approach to tune binding affinities. In combination with the nIR fluorescence properties of SWCNTs, these new hybrid materials promise many applications in integrin targeting and bioimaging.
The large gap in time scales between membrane fusion occurring in biological systems during neurotransmitter release and fusion observed between model membranes has provoked speculations over a large number of possible factors that might explain this discrepancy. One possible reason is an elevated lateral membrane tension present in the presynaptic membrane. We investigated the tension-dependency of fusion using model membranes equipped with a minimal fusion machinery consisting of syntaxin 1, synaptobrevin and SNAP 25. Two different strategies were realized; one based on supported bilayers and the other one employing sessile giant liposomes. In the first approach, isolated patches of planar bilayers derived from giant unilamellar vesicles containing syntaxin 1 and preassembled SNAP 25 (ΔN-complex) were deposited on a dilatable PDMS sheet. In a second approach, lateral membrane tension was controlled through the adhesion of intact giant unilamellar vesicles on a functionalized surface. In both approaches fusion efficiency increases considerably with lateral tension and we identified a threshold tension of 3.4 mN m−1, at which the number of fusion events is increased substantially.
Neurotransmitters are an important class of messenger molecules. They govern chemical communication between cells for example in the brain. The spatiotemporal propagation of these chemical signals is a crucial part of communication between cells. Thus, the spatial aspect of neurotransmitter release is equally important as the mere time-resolved measurement of these substances. In conclusion, without tools that provide the necessary spatiotemporal resolution, chemical signaling via neurotransmitters cannot be studied in greater detail. In this review article we provide a critical overview about sensors/probes that are able to monitor neurotransmitters. Our focus are sensing concepts that provide or could in the future provide the spatiotemporal resolution that is necessary to 'image' dynamic changes of neurotransmitter concentrations around cells. These requirements set the bar for the type of sensors we discuss. The sensor must be small enough (if possible on the nanoscale) to provide the envisioned spatial resolution and it should allow parallel (spatial) detection. In this article we discuss both optical and electrochemical concepts that meet these criteria. We cover techniques that are based on fluorescent building blocks such as nanomaterials, proteins and organic dyes. Additionally, we review electrochemical array techniques and assess limitations and possible future directions.
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