Fluorescent Single-walled Carbon Nanotubes Research Articles

(Invited) Near Infrared Imaging of Cellular Signaling

Cells use molecules to exchange information. A very prominent example is neurotransmitter signaling between neuronal cells or paracrine signaling between blood cells or bacteria. However, it is up to date difficult to visualize signaling processes between cells with high spatial, temporal and chemical resolution. We are interested in developing fluorescent sensors/probes that overcome these limitations and answer novel biological questions.For that purpose we use near infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs). SWCNTs fluoresce in the nIR tissue transparency window (900 nm -1700 nm) and do not bleach. They are chemically modified to render them selective for biological signaling molecules. In this context, we developed different chemical strategies that include screening for artificial binding motifs, aptamers and nanobodies. Using these strategies we created sensors for the neurotransmitters dopamine/serotonin, reactive oxygen species and bacterial signaling molecule. These sensors change their fluorescence in the presence of the target molecule and detect down to single molecules. Fast parallel imaging of many of those sensors provides a NIR image (>900 nm) of the neurotransmitter concentration around cells. This technique was used to image hot spots of dopamine release from primary dopaminergic under electrical stimulation and assess the heterogeneity of release sites. Neurotransmitters are not only relevant in the neuronal system but as well in the periphery e.g. in blood cells. In this context, we imaged for the first time how human blood platelets release serotonin after activation and resolved the location and the time scale of release. Finally, such sensors resolve signaling between bacteria and even detect them. These results illustrate that tailored SWCNTs are powerful tools for quantitative chemical imaging of biological processes.

Monitoring Plant Health with Near-Infrared Fluorescent H2O2 Nanosensors

Near-infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) were designed and interfaced with leaves of Arabidopsis thaliana plants to report hydrogen peroxide (H2O2), a key signaling molecule associated with the onset of plant stress. The sensor nIR fluorescence response (>900 nm) is quenched by H2O2 with selectivity against other stress-associated signaling molecules and within the plant physiological range (10−100 H2O2 μM). In vivo remote nIR imaging of H2O2 sensors enabled optical monitoring of plant health in response to stresses including UV-B light (−11%), high light (−6%), and a pathogen-related peptide (flg22) (−10%), but not mechanical leaf wounding (<3%). The sensor’s high biocompatibility was reflected on similar leaf cell death (<5%) and photosynthetic rates to controls without SWCNT. These optical nanosensors report early signs of stress and will improve our understanding of plant stress communication, provide novel tools for precision agriculture, and optimize the use of agrochemicals in the environment.

(Invited) A Wearable Optical Microfibrous Biomaterial with Encapsulated Nanosensors Enables Wireless Monitoring of Oxidative Stress

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 which 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 wounds. The environmentally sensitive and non-photobleachable fluorescence of SWCNTs enables continuous analyte monitoring without a 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 can be utilized to spatially resolve peroxide concentrations on a wound surface using a camera and can be integrated into commercial wound bandages without being altered or losing their optical properties.

Quantification of Nitric Oxide Concentration Using Single-Walled Carbon Nanotube Sensors.

Nitric oxide (NO), a free radical present in biological systems, can have many detrimental effects on the body, from inflammation to cancer. Due to NO’s short half-life, detection and quantification is difficult. The inability to quantify NO has hindered researchers’ understanding of its impact in healthy and diseased conditions. Single-walled carbon nanotubes (SWNTs), when wrapped in a specific single-stranded DNA chain, becomes selective to NO, creating a fluorescence sensor. Unfortunately, the correlation between NO concentration and the SWNT’s fluorescence intensity has been difficult to determine due to an inability to immobilize the sensor without altering its properties. Through the use of a recently developed sensor platform, systematic studies can now be conducted to determine the correlation between SWNT fluorescence and NO concentration. This paper explains the methods used to determine the equations that can be used to convert SWNT fluorescence into NO concentration. Through the use of the equations developed in this paper, an easy method for NO quantification is provided. The methods outlined in this paper will also enable researchers to develop equations to determine the concentration of other reactive species through the use of SWNT sensors.

A Wearable Optical Microfibrous Biomaterial with Encapsulated Nanosensors Enables Wireless Monitoring of Oxidative Stress

AbstractIn 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.

Analysis of Single-Walled Carbon Nanotubes in Estuarine Sediments by Density Gradient Ultracentrifugation Coupled to Near-Infrared Fluorescence Spectroscopy Reveals Disassociation of Residual Metal Catalyst Nanoparticles.

The continued growth of the nanotechnology industry and the incorporation of nanomaterials into consumer applications will inevitably lead to their release into environmental systems. Single-walled carbon nanotubes (SWCNTs) in particular have exhibited many attractive optical, mechanical, and electrical properties that lend themselves to new and exciting applications. Assessing their environmental impact upon release into the environment is contingent upon quantifying and characterizing SWCNTs in environmental matrixes. In this study, SWCNTs were isolated from estuarine sediments using density gradient ultracentrifugation (DGU), followed by online flow-through analysis of the density fractions via near-infrared spectroscopy. This approach yielded significant improvements in the quantitative detection limit, from 62 to 1.5 μg g-1. In addition, fractions of the density gradient were also obtained for further analysis by bulk inductively coupled plasma mass spectrometry (ICP-MS) and single-particle ICP-MS. Using fluorescent, semiconductive SWCNTs, the primary fluorescent nanotube fraction was found to be separated from the sediment matrix during DGU; however, the residual metal catalyst particles that had been assumed to be physically bound to the SWCNTs were found to form a separate band in the density gradient apart from the fluorescent SWCNTs. This result was repeated for a number of SWCNT types regardless of the metal catalyst and synthesis method, with a 0.1 g cm-3 density difference between most fractions. The apparent disconnect between the fluorescent fraction of SWCNTs and their metal-containing constituents potentially complicates CNT risk assessment as analysis techniques focusing solely on either CNT fluorescence or metal fingerprints may misrepresent exposure concentrations and their toxicological implications.

(Invited) In Vivo Analyte Detection Via Single-Walled Carbon Nanotube Near-Infrared Fluorescence

Implantable optical nanosensors hold the promise of rapid, minimally-invasive longitudal monitoring of analytes in a patient or animal. Such devices will allow for informed clinical intervention based on changes in drug, biomarker, or disease state profiles. We developed implantable nanosensors based on the optical properties of single-walled carbon nanotubes (SWCNT). SWCNT fluorescence is in the ‘tissue-transparent’ near-infrared spectrum, allowing for fluorescence measurements after implantation inside an animal. Further, SWCNT fluorescence is photostable, allowing for multiple measurements over time. We engineered separate SWCNT-based sensors such that their fluorescence emission responded to various analytes, including small molecule drugs as well as protein and nucleic acid biomarkers. Each sensor device was immobilized into a semi-permeable biocompatible membrane, allowing analyte penetration but retaining SWCNT inside. We implanted each sensor device into live mice, either in the peritoneal cavity or subcutaneously, and performed whole-mouse near-infrared fluorescence imaging to ensure the stability of the membrane and sensor brightness. We used a sensor implant specific for the chemotherapeutic doxorubicin to drug monitor transport within the animal. We used sensor implants specific for the ovarian cancer protein biomarker HE4 or for individual microRNA sequences to measure exogenous or cancer-generated quantities of each biomarker in mice. We expect these implantable sensors to have potential as research tools or clinical diagnostics to monitor pharmacokinetics or locally measure disease biomarkers in real-time.

Dye Quenching of Carbon Nanotube Fluorescence Reveals Structure-Selective Coating Coverage.

Many properties and applications of single-wall carbon nanotubes (SWCNTs) depend strongly on the coatings that allow their suspension in aqueous media. We report that SWCNT fluorescence is quenched by reversible physisorption of dye molecules such as methylene blue, and that measurements of that quenching can be used to infer structure-specific exposures of the nanotube surface to the surrounding solution. SWCNTs suspended in single-stranded DNA oligomers show quenching dependent on the combination of nanotube structure and ssDNA base sequence. Several sequences are found to give notably high or low surface coverages for specific SWCNT species. These effects seem correlated with the selective recognitions used for DNA-based structural sorting of nanotubes. One notable example is that dye quenching of fluorescence from SWCNTs coated with the (ATT)4 base sequence is far stronger for one (7,5) enantiomer than for the other, showing that coating coverage is associated with the coating affinity difference reported previously for this system. Equilibrium modeling of quenching data has been used to extract parameters for comparative complexation constants and accessible surface areas. Further insights are obtained from molecular dynamics simulations, which give estimated contact areas between ssDNA and SWCNTs that correlate with experimentally inferred surface exposures and account for the enantiomeric discrimination of (ATT)4.

A Fiber Optic Interface Coupled to Nanosensors: Applications to Protein Aggregation and Organic Molecule Quantification.

Fluorescent nanosensors hold promise to address analytical challenges in the biopharmaceutical industry. The monitoring of therapeutic protein critical quality attributes such as aggregation is a long-standing challenge requiring low detection limits and multiplexing of different product parameters. However, general approaches for interfacing nanosensors to the biopharmaceutical process remain minimally explored to date. Herein, we design and fabricate a integrated fiber optic nanosensor element, measuring sensitivity, response time, and stability for applications to the rapid process monitoring. The fiber optic-nanosensor interface, or optode, consists of label-free nIR fluorescent single-walled carbon nanotube transducers embedded within a protective yet porous hydrogel attached to the end of the fiber waveguide. The optode platform is shown to be capable of differentiating the aggregation status of human immunoglobulin G, reporting the relative fraction of monomers and dimer aggregates with sizes 5.6 and 9.6 nm, respectively, in under 5 min of analysis time. We introduce a lab-on-fiber design with potential for at-line monitoring with integration of 3D-printed miniaturized sensor tips having high mechanical flexibility. A parallel measurement of fluctuations in laser excitation allows for intensity normalization and significantly lower noise level (3.7 times improved) when using lower quality lasers, improving the cost effectiveness of the platform. As an application, we demonstrate the capability of the fully integrated lab-on-fiber system to rapidly monitor various bioanalytes including serotonin, norepinephrine, adrenaline, and hydrogen peroxide, in addition to proteins and their aggregation states. These results in total constitute an effective form factor for nanosensor-based transducers for applications in industrial process monitoring.

Collecting up to 115% of Singlet-Fission Products by Single-Walled Carbon Nanotubes.

In this contribution, we focused on integrating a phenylene-bridged dibenzodiazahexacene dimer (o-DAD), which is singlet fission (SF) active, onto single-walled carbon nanotubes (SWCNTs) as a low-energy sink for energetically low lying excited states that stem from SF. Spectroscopic and microscopic assays assisted in documenting that SWCNT/o-DAD feature high stability in THF as a result of electronic interactions between the individual constituents. For example, statistical Raman analysis underlined n-doping of SWCNTs in the presence of o-DAD. Fluorescence spectroscopy prompted an energy transfer between the individual constituents, a conclusion that was exclusively derived from the quenching of the o-DAD-centered fluorescence. Excitation spectroscopy with a focus on the SWCNT fluorescence confirmed independently this conclusion by showing o-DAD-centered features. Our work was rounded off by time-resolved transient absorption measurements with SWCNT/o-DAD, in which evidence was gathered for the sequential o-DAD-centered SF with an efficiency of 112% followed by a unidirectional energy transfer from o-DAD to SWCNT and a rapid deactivation. The energy transfer efficiency from SF products such as (S1S0)CT and 1(T1T1) exceeded the 100% threshold with values of 115%, which is conventionally found in energy transfer schemes.

(Invited) In Vivo Analyte Detection Via Single-Walled Carbon Nanotube Near-Infrared Fluorescence

Implantable optical nanosensors hold the promise of rapid, minimally-invasive longitudal monitoring of analytes in a patient or animal. Such devices will allow for informed clinical intervention based on changes in drug, biomarker, or disease state profiles. We developed implantable nanosensors based on the optical properties of single-walled carbon nanotubes (SWCNT). SWCNT fluorescence is in the ‘tissue-transparent’ near-infrared spectrum, allowing for fluorescence measurements after implantation inside an animal. Further, SWCNT fluorescence is photostable, allowing for multiple measurements over time. We engineered separate SWCNT-based sensors such that their fluorescence emission responded to various analytes, including small molecule drugs as well as protein and nucleic acid biomarkers. Each sensor device was immobilized into a semi-permeable biocompatible membrane, allowing analyte penetration but retaining SWCNT inside. We implanted each sensor device into live mice, either in the peritoneal cavity or subcutaneously, and performed whole-mouse near-infrared fluorescence imaging to ensure the stability of the membrane and sensor brightness. We used a sensor implant specific for the chemotherapeutic doxorubicin to drug monitor transport within the animal. We used sensor implants specific for the ovarian cancer protein biomarker HE4 or for individual microRNA sequences to measure exogenous or cancer-generated quantities of each biomarker in mice. We expect these implantable sensors to have potential as research tools or clinical diagnostics to monitor pharmacokinetics or locally measure disease biomarkers in real-time.

The Impact of Covalent Functionalization on Single-Walled Carbon Nanotube Biosensor Fluorescence and Function

Single-walled carbon nanotubes (SWCNTs) are advantageous signal transduction elements for biological imaging and sensing due to their notable fluorescence in the near-infrared (NIR) region with minimal light absorbance by water or scattering by tissue (1). Recently, exciting developments in SWCNT surface chemistry have shown the retention and improvement of fluorescence intensity in SWCNTs after covalent surface functionalization (2). These chemistries provide chemical handles for specific attachment of molecules of interest to the SWCNT surface while keeping the intrinsic SWCNT NIR fluorescence intact for bioimaging and sensing applications. However, despite their unperturbed fluorescence, it is unclear whether functionalized SWCNTs maintain their ability for molecular sensing, which necessitates a modulation in SWCNT fluorescence provided by the molecular recognition of a noncovalently surface-adsorbed polymer. We compare the photophysical properties of functionalized SWCNTs to their pristine counterparts, and show SWCNT optical properties remain available for sensing applications, however with noted attenuation of fluorescence modulation between certain surface coating and analyte pairs. Molecular recognition provided by phospholipid SWCNT surface coatings maintain their ability to detect molecular analytes such as fibrinogen and insulin. Conversely, DNA oligonucleotide surface coatings under-perform for molecular recognition of dopamine compared to sensors constructed from pristine SWCNTs. We also discuss the use of covalent handles to tune the intrinsic properties of the SWCNT surface for use as biological tools. Lastly, we explore the application of covalently functionalized SWCNTs as dual-functional nanoparticles with both targeting and sensing capabilities. Our work establishes the potential advantages and drawbacks of covalent SWCNT functionalization, despite preservation of SWCNT fluorescence, and implications for applications in molecular sensing.(1) Bonis-O’Donnell, J. T. D.; Page, R. H.; Beyene, A. G.; Tindall, E. G.; McFarlane, I. R.; Landry, M. P. Dual Near-Infrared Two-Photon Microscopy for Deep-Tissue Dopamine Nanosensor Imaging. Adv. Funct. Mater. 2017, 27 (39), 1–10.(2) Setaro, A.; Adeli, M.; Glaeske, M.; Przyrembel, D.; Bisswanger, T.; Gordeev, G.; Maschietto, F.; Faghani, A.; Paulus, B.; Weinelt, M.; et al. Preserving π-Conjugation in Covalently Functionalized Carbon Nanotubes for Optoelectronic Applications. Nat. Commun. 2017, 8, 1–7.

Near-Infrared Imaging of Serotonin Release from Cells with Fluorescent Nanosensors

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 single-walled 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.

(Invited) Near Infrared Imaging of Chemical Signaling between Cells Using Fluorescent Nanosensors

Cells use small molecules to exchange information. A very prominent example is neurotransmitter signaling between neuronal cells or paracrine signaling between blood cells. However, it is up to date difficult to visualize signaling processes between cells with high spatial, temporal and chemical resolution. We are interested in developing fluorescent sensors/probes that overcome these limitations and answer novel biological questions.For that purpose we use near infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs). SWCNTs fluoresce in the nIR tissue transparency window (900 nm -1700 nm) and do not bleach. They are chemically modified to render them selective for neurotransmitters such as dopamine and serotonin. In this context, we developed different chemical strategies that include screening for artificial binding motifs, aptamers and nanobodies. Using these strategies we created sensors for the neurotransmitters dopamine and serotonin that increase their fluorescence in the presence of the target molecule and detect down to single molecules. Fast parallel imaging of many of those sensors provides a nIR image (>900 nm) of the neurotransmitter concentration around cells. This technique was used to image hot spots of dopamine release from primary dopaminergic neurons. It enabled us to observe localized release sites and correlate it with the exocytosis machinery of the cells. Neurotransmitters are not only relevant in the neuronal system but as well in the periphery e.g. in blood cells. In this context, we imaged for the first time how human blood platelets release serotonin after activation and resolved the heterogeneity in release patterns. These results illustrate that tailored SWCNTs are powerful tools for quantitative chemical imaging of biological processes.

Covalent Surface Modification Effects on Single‐Walled Carbon Nanotubes for Targeted Sensing and Optical Imaging

AbstractOptical nanoscale technologies often implement covalent or noncovalent strategies for the modification of nanoparticles, whereby both functionalizations are leveraged for multimodal applications but can affect the intrinsic fluorescence of nanoparticles. Specifically, single‐walled carbon nanotubes (SWCNTs) can enable real‐time imaging and cellular delivery; however, the introduction of covalent SWCNT sidewall functionalizations often attenuates SWCNT fluorescence. Recent advances in SWCNT covalent functionalization chemistries preserve the SWCNT's pristine graphitic lattice and intrinsic fluorescence, and here, such covalently functionalized SWCNTs maintain intrinsic fluorescence‐based molecular recognition of neurotransmitter and protein analytes. The covalently modified SWCNT nanosensor preserves its fluorescence response towards its analyte for certain nanosensors, presumably dependent on the intermolecular interactions between SWCNTs or the steric hindrance introduced by the covalent functionalization that hinders noncovalent interactions with the SWCNT surface. These SWCNT nanosensors are further functionalized via their covalent handles with a targeting ligand, biotin, to self‐assemble on passivated microscopy slides, and these dual‐functionalized SWCNT materials are explored for future use in multiplexed sensing and imaging applications.

Monitoring Plant Health with Near-Infrared Fluorescent H2O2 Nanosensors.

Near-infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) were designed and interfaced with leaves of Arabidopsis thaliana plants to report hydrogen peroxide (H2O2), a key signaling molecule associated with the onset of plant stress. The sensor nIR fluorescence response (>900 nm) is quenched by H2O2 with selectivity against other stress-associated signaling molecules and within the plant physiological range (10-100 H2O2 μM). In vivo remote nIR imaging of H2O2 sensors enabled optical monitoring of plant health in response to stresses including UV-B light (-11%), high light (-6%), and a pathogen-related peptide (flg22) (-10%), but not mechanical leaf wounding (<3%). The sensor's high biocompatibility was reflected on similar leaf cell death (<5%) and photosynthetic rates to controls without SWCNT. These optical nanosensors report early signs of stress and will improve our understanding of plant stress communication, provide novel tools for precision agriculture, and optimize the use of agrochemicals in the environment.

Single-Walled Carbon Nanotube-Assisted Antibiotic Delivery and Imaging in S. epidermidis Strains Addressing Antibiotic Resistance.

Although conventional antibiotics have evolved as a staple of modern medicine, increasing antibiotic resistance and the lack of antibiotic efficacy against new bacterial threats is becoming a major medical threat. In this work, we employ single-walled carbon nanotubes (SWCNTs) known to deliver and track therapeutics in mammalian cells via intrinsic near-infrared fluorescence as carriers enhancing antibacterial delivery of doxycycline and methicillin. SWCNTs dispersed in water by antibiotics without the use of toxic bile salt surfactants facilitate efficacy enhancement for both antibiotics against Staphylococcus epidermidis strain showing minimal sensitivity to methicillin. Doxycycline to which the strain did not show resistance in complex with SWCNTs provides only minor increase in efficacy, whereas the SWCNTs/methicillin complex yields up to 40-fold efficacy enhancement over antibiotics alone, suggesting that SWCNT-assisted delivery may circumvent antibiotic resistance in that bacterial strain. At the same time SWCNT/antibiotic formulations appear to be less toxic to mammalian cells than antibiotics alone suggesting that nanomaterial platforms may not restrict potential biomedical applications. The improvement in antibacterial performance with SWCNT delivery is tested via 3 independent assays—colony count, MIC (Minimal Inhibitory Concentration) turbidity and disk diffusion, with the statistical significance of the latter verified by ANOVA and Dunnett’s method. The potential mechanism of action is attributed to SWCNT interactions with bacterial cell wall and adherence to the membrane, as substantial association of SWCNT with bacteria is observed—the near-infrared fluorescence microscopy of treated bacteria shows localization of SWCNT fluorescence in bacterial clusters, scanning electron microscopy verifies SWCNT association with bacterial surface, whereas transmission electron microscopy shows individual SWCNT penetration into bacterial cell wall. This work characterizes SWCNTs as novel advantageous antibiotic delivery/imaging agents having the potential to address antibiotic resistance.

Near-Infrared Imaging of Serotonin Release from Cells with Fluorescent Nanosensors.

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 single-walled 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.

Multi-Drug/Gene NASH Therapy Delivery and Selective Hyperspectral NIR Imaging Using Chirality-Sorted Single-Walled Carbon Nanotubes.

Single-walled carbon nanotubes (SWCNTs) can serve as drug delivery/biological imaging agents, as they exhibit intrinsic fluorescence in the near-infrared, allowing for deeper tissue imaging while providing therapeutic transport. In this work, CoMoCAT (Cobalt Molybdenum Catalyst) SWCNTs, chirality-sorted by aqueous two-phase extraction, are utilized for the first time to deliver a drug/gene combination therapy and image each therapeutic component separately via chirality-specific SWCNT fluorescence. Each of (7,5) and (7,6) sorted SWCNTs were non-covalently loaded with their specific payload: the PI3 kinase inhibitor targeting liver fibrosis or CCR5 siRNA targeting inflammatory pathways with the goal of addressing these processes in nonalcoholic steatohepatitis (NASH), ultimately to prevent its progression to hepatocellular carcinoma. PX-866-(7,5) SWCNTs and siRNA-(7,6) SWCNTs were each imaged via characteristic SWCNT emission at 1024/1120 nm in HepG2 and HeLa cells by hyperspectral fluorescence microscopy. Wavelength-resolved imaging verified the intracellular transport of each SWCNT chirality and drug release. The therapeutic efficacy of each formulation was further demonstrated by the dose-dependent cytotoxicity of SWCNT-bound PX-866 and >90% knockdown of CCR5 expression with SWCNT/siRNA transfection. This study verifies the feasibility of utilizing chirality-sorted SWCNTs for the delivery and component-specific imaging of combination therapies, also suggesting a novel nanotherapeutic approach for addressing the progressions of NASH to hepatocellular carcinoma.

An in Vivo Nanosensor Measures Compartmental Doxorubicin Exposure.

Preclinical measurements of drug exposure to specific organs and tissues is normally performed by destructive methods. Tissue-specific measurements are important, especially for drugs with intractable dose-limiting toxicities, such as doxorubicin-mediated cardiotoxicity. We developed a method to rapidly quantify doxorubicin exposure to tissues within living organisms using an implantable optical nanosensor that can be interrogated noninvasively following surgical implantation. The near-infrared fluorescence of single-walled carbon nanotubes functionalized with DNA was found to respond to doxorubicin via a large and uniform red-shift. We found this to be common to DNA-intercalating agents, including anthracycline compounds such as doxorubicin. Doxorubicin was measured in buffer and serum, intracellularly, and from single nanotubes on a surface. Doxorubicin adsorption to the DNA-suspended nanotubes did not displace DNA but bound irreversibly. We incorporated the nanosensors into an implantable membrane which allowed cumulative detection of doxorubicin exposure in vivo. On implanting the devices into different compartments, such as subcutaneously and within the peritoneal cavity, we achieved real-time, minimally invasive detection of doxorubicin injected into the peritoneal cavity, as well as compartment-specific measurements. We measured doxorubicin translocation across the peritoneal membrane in vivo. Robust, minimally invasive pharmacokinetic measurements in vivo suggest the suitability of this technology for preclinical drug discovery applications.