Noncovalent hybrids of single-stranded DNA and single-walled carbon nanotubes (SWCNTs) have demonstrated applications in biomedical imaging and sensing due to their enhanced biocompatibility and photostable, environmentally responsive near-infrared (NIR) fluorescence. The fundamental properties of such DNA-SWCNTs have been studied to determine the correlative relationships between oligonucleotide sequence and length, SWCNT species, and the physical attributes of the resultant hybrids. However, intracellular environments introduce harsh conditions that can change the physical identities of the hybrid nanomaterials, thus altering their intrinsic optical properties. Here, through visible and NIR fluorescence imaging in addition to confocal Raman microscopy, we show that the oligonucleotide length controls the relative uptake, intracellular optical stability, and retention of DNA-SWCNTs in mammalian cells. Although the absolute NIR fluorescence intensity of DNA-SWCNTs in murine macrophages increases with increasing oligonucleotide length (from 12 to 60 nucleotides), we found that shorter oligonucleotide DNA-SWCNTs undergo a greater magnitude of spectral shift and are more rapidly internalized and expelled from the cell after 24 h. Furthermore, by labeling the DNA with a fluorophore that dequenches upon removal from the SWCNT surface, we found that shorter oligonucleotide strands are displaced from the SWCNT within the cell, altering the physical identity and changing the fate of the internalized nanomaterial. Finally, through a pharmacological inhibition study, we identified the mechanism of SWCNT expulsion from the cells as lysosomal exocytosis. These findings provide a fundamental understanding of the interactions between SWCNTs and live cells as well as evidence suggesting the ability to control the biological fate of the nanomaterials merely by varying the type of DNA wrapping.

In an effort to facilitate personalized medical approaches, the continuous and noninvasive monitoring of biochemical information using wearable technologies can enable a detailed understanding of an individual's physiology. Reactive oxygen species (ROS) are a class of oxygen‐containing free radicals that function in a wide range of biological processes. In wound healing applications, the continuous monitoring of ROS through a wearable diagnostics platform is essential for the prevention of chronicity and pathogenic infection. Here, a versatile one‐step procedure is utilized to fabricate optical core‐shell microfibrous textiles incorporating single‐walled carbon nanotubes (SWCNTs) for the real‐time optical monitoring of hydrogen peroxide concentrations in in vitro wounds. The environmentally sensitive and non‐photobleachable fluorescence of SWCNTs enables continuous analyte monitoring without decay in signal over time. The existence of multiple chiralities of SWCNTs emitting near‐infrared fluorescence with narrow bandwidths allows a ratiometric signal readout invariant to the excitation source distance and exposure time. The individual fibers encapsulate the SWCNT nanosensors for at least 21 days without apparent loss in structural integrity. Moreover, the microfibrous textiles are utilized to spatially resolve peroxide concentrations using a camera and further integrated into commercial wound bandages without significant degradation in their optical properties.

Single-walled carbon nanotubes (SWCNTs) functionalized with short single-stranded DNA have been extensively studied within the last decade for biomedical applications due to the high dispersion efficiency and intrinsic biocompatibility of DNA as well as the photostable and tunable fluorescence of SWCNTs. Characterization of their physical properties, particularly their length distribution, is of great importance regarding their application as a bioengineered research tool and clinical diagnostic agent. Conventionally, atomic force microscopy (AFM) has been used to quantify the length of DNA-SWCNTs by depositing the hybrids onto an electrostatically charged flat surface. Here, we demonstrate that hybrids of DNA-SWCNTs with different oligomeric DNA sequences ((GT)6 and (GT)30) differentially deposit on the AFM substrate, resulting in significant inaccuracies in the reported length distributions of the parent solutions. Using a solution-based surfactant exchange technique, we placed both samples into a common surfactant wrapping and found identical SWCNT length distributions upon surface deposition. Additionally, by spincoating the surfactant wrapped SWCNTs onto a substrate, thus mitigating effects of electrostatic interactions, we found length distributions that did not depend on DNA sequence but were significantly longer than electrostatic deposition methods, illuminating the inherent bias of the surface deposition method. Quantifying the coverage of DNA molecules on each SWCNT through both absorbance spectroscopy and direct observation, we found that the density of DNA per SWCNT was significantly higher in short (GT)6-SWCNTs (length < 100 nm) compared to long (GT)6-SWCNTs (length > 100 nm). In contrast, we found no dependence of the DNA density on SWCNT length in (GT)30-SWCNT hybrids. Thus, we attribute differences in the observed length distributions of DNA-SWCNTs to variations in electrostatic repulsion induced by sequencedependent DNA density.

Intracellular vesicle trafficking involves a complex series of biological pathways used to sort, recycle, and degrade extracellular components, including engineered nanomaterials (ENMs) which gain cellular entry via active endocytic processes. A recent emphasis on routes of ENM uptake has established key physicochemical properties which direct certain mechanisms, yet relatively few studies have identified their effect on intracellular trafficking processes past entry and initial subcellular localization. Here, we developed and applied an approach where single-walled carbon nanotubes (SWCNTs) play a dual rolethat of an ENM undergoing intracellular processing, in addition to functioning as the signal transduction element reporting these events in individual cells with single organelle resolution. We used the exceptional optical properties exhibited by noncovalent hybrids of single-stranded DNA and SWCNTs (DNA−SWCNTs) to report the progression of intracellular processing events via two orthogonal hyperspectral imaging approaches of near-infrared (NIR) fluorescence and resonance Raman scattering. A positive correlation between fluorescence and G-band intensities was uncovered within single cells, while exciton energy transfer and eventual aggregation of DNA−SWCNTs were observed to scale with increasing time after internalization. An analysis pipeline was developed to colocalize and deconvolute the fluorescence and Raman spectra of subcellular regions of interest (ROIs), allowing for singlechirality component spectra to be obtained with submicron spatial resolution. This approach uncovered correlations between DNA−SWCNT concentration, dielectric modulation, and irreversible aggregation within single intracellular vesicles. An immunofluorescence assay was designed to directly observe the DNA−SWCNTs in labeled endosomal vesicles, revealing a distinct relationship between the physical state of organelle-bound DNA−SWCNTs and the dynamic luminal conditions during endosomal maturation processes. Finally, we trained a machine learning algorithm to predict endosome type using the Raman spectra of the vesicle-bound DNA−SWCNTs, enabling major components in the endocytic pathway to be simultaneously visualized using a single intracellular reporter.

Single-walled carbon nanotubes (SWCNTs) have been used in a variety of sensing and imaging applications over the past few years due to their unique optical properties. In the solution phase, SWCNTs are employed as near-infrared (NIR) fluorescence-based sensors of target analytes via modulations in emission intensity and/or wavelength. In an effort to lower the limit of detection, research has been conducted into isolating SWCNTs adhered to surfaces for potential single molecule analyte detection. However, it is known that SWCNT fluorescence is adversely affected by the inherently rough surfaces that are conventionally used for their observation (e.g., glass coverslip), potentially interfering with fluorescence-based analyte detection. Here, using a spin-coating method with thin films of alginate and SWCNTs, we demonstrate that a novel hydrogel platform can be created to investigate immobilized individual SWCNTs without significantly perturbing their optical properties as compared to solution-phase values. In contrast to the glass coverslip, which red-shifted DNA-functionalized (6,5)-SWCNTs by an average of 3.4 nm, the hydrogel platform reported emission wavelengths that statistically matched the solution-phase values. Additionally, the heterogeneity in the wavelength measurements, as determined from the width of created histograms, was reduced nearly by a factor of 3 for the SWCNTs in the hydrogel platform when compared to glass coverslips. Using long SWCNTs, i.e., those with an average length above the diffraction limit of our microscope, we show that a glass coverslip can induce optical heterogeneity along the length of a single SWCNT regardless of its surface functionalization. This is again significantly mitigated when examining the long SWCNTs in the hydrogel platform. Finally, we show that upon the addition of a model analyte (calcium chloride), the optical response can be spatially resolved along the length of a single SWCNT, enabling localized analyte detection on the surface of a single nanoscale sensor.