RESUMO
Single-walled carbon nanotubes (SWCNTs) are versatile near infrared (NIR) fluorescent building blocks for biosensors. Their surface is chemically tailored to respond to analytes by a change in fluorescence. However, intensity-based signals are easily affected by external factors such as sample movements. Here, we demonstrate fluorescence lifetime imaging microscopy (FLIM) of SWCNT-based sensors in the NIR. We tailor a confocal laser scanning microscope (CLSM) for NIR signals (>800â nm) and employ time correlated single photon counting of (GT)10 -DNA functionalized SWCNTs. They act as sensors for the important neurotransmitter dopamine. Their fluorescence lifetime (>900â nm) decays biexponentially and the longer lifetime component (370â ps) increases by up to 25 % with dopamine concentration. These sensors serve as paint to cover cells and report extracellular dopamine in 3D via FLIM. Therefore, we demonstrate the potential of fluorescence lifetime as a readout of SWCNT-based NIR sensors.
Assuntos
Nanotubos de Carbono , Fluorescência , Nanotubos de Carbono/química , Dopamina , Microscopia de Fluorescência/métodos , Corantes Fluorescentes/químicaRESUMO
Single wall carbon nanotubes (SWCNTs) functionalized with (bio-)polymers such as DNA are soluble in water and sense analytes by analyte-specific changes of their intrinsic fluorescence. Such SWCNT-based (bio-)sensors translate the binding of a molecule (molecular recognition) into a measurable optical signal. This signal transduction is crucial for all types of molecular sensors to achieve high sensitivities. Although there is an increasing number of SWCNT-based sensors, there is yet no molecular understanding of the observed changes in the SWCNT's fluorescence. Here, we report THz experiments that map changes in the local hydration of the solvated SWCNT upon binding of analytes such as the neurotransmitter dopamine or the vitamin riboflavin. The THz amplitude signal serves as a measure of the coupling of charge fluctuations in the SWCNTs to the charge density fluctuations in the hydration layer. We find a linear (inverse) correlation between changes in THz amplitude and the intensity of the change in fluorescence induced by the analytes. Simulations show that the organic corona shapes the local water, which determines the exciton dynamics. Thus, THz signals are a quantitative predictor for signal transduction strength and can be used as a guiding chemical design principle for optimizing fluorescent biosensors.
RESUMO
Neurotransmitters are released by neuronal cells to exchange information. Resolving their spatiotemporal patterns is crucial to understand chemical neurotransmission. Here, we present a ratiometric sensor for the neurotransmitter dopamine that combines Egyptian blue (CaCuSi4O10) nanosheets (EB-NS) and single-walled carbon nanotubes (SWCNTs). They both fluoresce in the near infrared (NIR) region, which is beneficial due to their ultra-low background and phototoxicity. (GT)10-DNA-functionalized monochiral (6,5)-SWCNTs increase their fluorescence (1000 nm) in response to dopamine, while EB-NS serve as a stable reference (936 nm). A robust ratiometric imaging scheme is implemented by directing these signals on two different NIR sensitive cameras. Additionally, we demonstrate stability against mechanical perturbations and image dopamine release from differentiated dopaminergic Neuro 2a cells. Therefore, this technique enables robust ratiometric and non-invasive imaging of cellular responses.
RESUMO
Fluorescent single-wall carbon nanotubes (SWCNTs) are used as nanoscale biosensors in diverse applications. Selectivity is built in by noncovalent functionalization with polymers such as DNA. Recently, covalent functionalization was demonstrated by conjugating guanine bases of adsorbed DNA to the SWCNT surface as guanine quantum defects (g-defects). Here, we create g-defects in (GT)10-coated SWCNTs (Gd-SWCNTs) and explore how this affects molecular sensing. We vary the defect densities, which shifts the E11 fluorescence emission by 55 nm to a λmax of 1049 nm. Furthermore, the Stokes shift between absorption and emission maximum linearly increases with defect density by up to 27 nm. Gd-SWCNTs represent sensitive sensors and increase their fluorescence by >70% in response to the important neurotransmitter dopamine and decrease it by 93% in response to riboflavin. Additionally, the extent of cellular uptake of Gd-SWCNTs decreases. These results show how physiochemical properties change with g-defects and that Gd-SWCNTs constitute a versatile optical biosensor platform.
Assuntos
Nanotubos de Carbono , DNA , Fluorescência , Nanotubos de Carbono/química , Guanina/química , Técnicas BiossensoriaisRESUMO
Single-wall carbon nanotubes (SWCNTs) are used in diverse applications that require chemical tailoring of the SWCNT surface, including optical sensing, imaging, targeted drug delivery and single-photon generation. SWCNTs have been noncovalently modified with (bio)polymers to preserve their intrinsic near-infrared fluorescence. However, demanding applications (e.g., requiring stability in biological fluids) would benefit from a stable covalent linkage between the SWCNT and the functional unit (e.g., antibody, fluorophore, drug). Here we present how to use diazonium salt chemistry to introduce sp3 quantum defects in the SWCNT carbon lattice to serve as handles for conjugation while preserving near-infrared fluorescence. In this protocol, we describe the straightforward, stable (covalent), highly versatile and scalable functionalization of SWCNTs with biomolecules such as peptides and proteins to yield near-infrared fluorescent SWCNT bioconjugates. We provide a step-by-step procedure covering SWCNT dispersion, quantum defect incorporation, bioconjugation, in situ peptide synthesis on SWCNTs, and characterization, which can be completed in 5-7 d.