Fluorescent Single-walled Carbon Nanotubes Research Articles

Differential near-infrared imaging of heterocysts using single-walled carbon nanotubes

The internalization of near-infrared (NIR) optical nanoprobes in photosynthetic microbes can be exploited for applications ranging from energy conversion to biomolecule delivery. However, the intrinsic, species-dependent properties of microbial cell walls, including their surface charge density, composition, thickness, and elasticity, can severely impact nanoprobe uptake and affect the cellular response. An examination of the interaction of the optical nanoprobe in various species and its impact on cell viability is, therefore, imperative for the development of new imaging technologies. Herein, we extend the technology recently developed for internalizing fluorescent single-walled carbon nanotubes (SWCNTs) in prokaryotes, specifically unicellular Synechocystis sp. PCC 6803, to a filamentous cyanobacterial strain, Nostoc punctiforme. Using a combination of NIR fluorescence, scanning electron microscopy (SEM), and Raman spectroscopy, we investigate uptake in vegetative cells as well as differentiated heterocysts. We demonstrate a strong dependence of long-term cell integrity, activity, and viability on SWCNT surface functionalization. We further show differential uptake of SWCNTs across a single filament, with positively charged functionalized SWCNTs preferentially localizing within the heterocysts of the filament. This cell dependency of the nanoparticle internalization motivates the use of SWCNTs as a NIR stain for monitoring cell differentiation.Graphical abstract

Carbon nanotube uptake in cyanobacteria for near-infrared imaging and enhanced bioelectricity generation in living photovoltaics.

The distinctive properties of single-walled carbon nanotubes (SWCNTs) have inspired the development of many novel applications in the field of cell nanobiotechnology. However, studies thus far have not explored the effect of SWCNT functionalization on transport across the cell walls of prokaryotes. We explore the uptake of SWCNTs in Gram-negative cyanobacteria and demonstrate a passive length-dependent and selective internalization of SWCNTs decorated with positively charged biomolecules. We show that lysozyme-coated SWCNTs spontaneously penetrate the cell walls of a unicellular strain and a multicellular strain. A custom-built spinning-disc confocal microscope was used to image the distinct near-infrared SWCNT fluorescence within the autofluorescent cells, revealing a highly inhomogeneous distribution of SWCNTs. Real-time near-infrared monitoring of cell growth and division reveal that the SWCNTs are inherited by daughter cells. Moreover, these nanobionic living cells retained photosynthetic activity and showed an improved photo-exoelectrogenicity when incorporated into bioelectrochemical devices.

(Invited) Real-Time Detection of Insulin Secreted By Pancreatic β-Cells Using Single-Walled Carbon Nanotube Sensors

Quantification of insulin is essential for diabetes research in general, and for the study of pancreatic β-cell function in particular. Fluorescent single-walled carbon nanotubes (SWCNTs) find many applications as optical nanosensors for biomolecules, proteins, and enzyme, with real-time feedback on target analyte binding1–4. Herein, fluorescent SWCNT are used for the recognition and real-time quantification of insulin5. Two approaches for rendering the SWCNT sensors for insulin are compared, using surface functionalization with either a natural insulin aptamer with known affinity to insulin, or a synthetic lipid-PEG, both of which show a modulation of the emitted fluorescence in response to insulin. Although the PEGylated-lipid has no prior affinity to insulin, its response to insulin is more stable and reproducible compared to the insulin aptamer-SWCNTs. The SWCNT sensors successfully detect and quantify insulin secreted by β-cells within the complex environment of the conditioned media, and the results are found to be in agreement with an enzyme-linked immunosorbent assay5. This novel analytical tool for real time quantification of insulin secreted by β-cells provides new opportunities for rapid assessment of β-cell function.(1) Bisker, G.; Dong, J.; Park, H. D.; Iverson, N. M.; Ahn, J.; Nelson, J. T.; Landry, M. P.; Kruss, S.; Strano, M. S. Protein-Targeted Corona Phase Molecular Recognition. Nat. Commun. 2016, 7, 1–14.(2) Bisker, G.; Bakh, N. A.; Lee, M. A.; Ahn, J.; Park, M.; O’Connell, E. B.; Iverson, N. M.; Strano, M. S. Insulin Detection Using a Corona Phase Molecular Recognition Site on Single-Walled Carbon Nanotubes. ACS Sensors 2018, 8 (2), 367–377.(3) Amir, D.; Hendler-Neumark, A.; Wulf, V.; Ehrlich, R.; Bisker, G. Oncometabolite Fingerprinting Using Fluorescent Single-Walled Carbon Nanotubes. Adv. Mater. Interfaces 2021.(4) Wulf, V.; Slor, G.; Rathee, P.; Amir, R. J.; Bisker, G. Dendron-Polymer Hybrids as Tailorable Coronae of Single-Walled Carbon Nanotube. ChemRxiv 2021. https://doi.org/10.26434/ChemRxiv.14452785.V1.(5) Ehrlich, R.; Hendler‐Neumark, A.; Wulf, V.; Amir, D.; Bisker, G. Optical Nanosensors for Real‐Time Feedback on Insulin Secretion by Β‐Cells. Small 2021, 17 (30), 2101660.

(Invited) Design of Robust Multiplexed Optical Nanosensors for In Vivo Disease Detection

The rapid, minimally-invasive detection of diverse analytes in vivo remains a goal for the diagnosis of cancer and related diseases. Toward this end, we are designing nanosensors based on the optical properties of single-walled carbon nanotubes (SWCNT) for use both in patients and at the bedside. SWCNT fluorescence is in the near-infrared window for biological imaging, is stable upon laser excitation, and responds rapidly to changes in its local environment. We are approaching the design of multiplexed implantable biomarker sensors through several routes, including developing novel bioconjugate chemistry approaches to antibodies and other molecular recognition elements to improve sensitivity of the sensors. We are also evaluating molecular tools to screen non-specific interactions within the local environment, improving the selectivity of these sensors. In parallel, we are using aqueous two-phase extraction (ATPE) methods to allow for separation of single-chirality SWCNT prior to bioconjugate chemistry approaches, allowing for specific detection of multiple analytes at once. Finally, we are exploring various hydrogel systems to allow for their immobilization upon injection into living systems and repeated monitoring of local biomarker concentrations. We expect these approaches to further bridge the translation of sensor devices toward clinical diagnostic tools. We hope to deploy SWCNT-based sensors to detect disease at early stages, evaluate disease response to treatment, and monitor disease progression. These aims will allow physicians to make rapid, informed decisions on patient prognosis and treatment.

Photoluminescence Brightening of Single-Walled Carbon Nanotubes Using Graphene Quantum Dots

Single-walled carbon nanotubes (SWCNTs) emit near-infrared (NIR) photoluminescence (PL) that spans the tissue transparency window, allowing them to optically penetrate biological tissue for deep-tissue optical sensing. Such optical SWCNT sensors are often functionalized with single stranded DNA (ssDNA) to promote biocompatibility, responsivity, and selectivity. However, the low brightness of these ssDNA-wrapped sensors restricts the depth at which such sensors can be implanted in biological tissue. In this work, we overcome this limitation by demonstrating the PL enhancement of ssDNA-wrapped SWCNTs through the incorporation of biocompatible graphene quantum dots (GQDs). We compare the enhancement effects of two preparations of GQDs, pristine (PGQDs) and nitrogen-doped (NGQDs) graphene quantum dots. Both preparations of GQDs were shown to significantly increase the fluorescence emissions of ssDNA-SWCNTs to a comparable extent after 3 h of incubation. We further observed a diameter dependence of this enhancement, with the larger (8,6) SWCNT chirality demonstrating a 30-fold enhancement as compared to the 2-fold enhancement observed for the (6,5) SWCNT chirality. This PL enhancement was shown to vary with the exposed surface area of the SWCNTs, whereby SWCNT chiralities with wrappings that yield larger exposed SWCNT surfaces resulted in increased PL enhancement on conjugation with the GQDs.Despite the similarities in the final fluorescence enhancements, the NGQDs and PGQDs showed kinetic differences in their brightening effects. The NGQDs were able to achieve stabilized SWCNT fluorescence brightening 2 min after their addition, whereas the SWCNTs showed continued PL increases even 1 h after the addition of the PGQDs. Electrochemical analysis of the NGQDs confirms their behavior as reducing agents, which have been previously shown to enhance the rate of SWCNT brightening through the passivation of oxygen defect sites. The brightening effects of the NGQDs were further demonstrated in confocal single-molecule measurements, where we observed a 3-fold increase in the detection of individual SWCNTs on conjugation to the NGQDs compared to the unconjugated ssDNA-SWCNTs. The photoluminescence enhancements enabled by the GQDs thus provides a promising tool for engineering the brightness of NIR sensors for biomedical applications and enabling improved detection in deep-tissue sensing and imaging applications.

Exploring the Role of Photosensitizer in Guanine Functionalization of Single-Wall Carbon Nanotubes

Single-walled carbon nanotubes (SWCNTs) have attracted a great deal of attention during the past two decades because of their unique optoelectronic properties and their potential applications in biomedical theranostics as well as nanoelectronics. It was recently discovered that it is possible to spatially modulate the exciton band gap of carbon nanotubes through a process known as guanine functionalization. In this reaction, the guanine nucleotides of an ssDNA strand wrapping a nanotube become covalently bonded to sidewall carbon atoms upon exposure to singlet oxygen.1 In the original work, the authors used xanthene derivatives such as rose bengal and erythrosine B as photosensitizers in order to generate singlet oxygen. It was assumed that this process was an independent step that did not involve the nanotube. However, we have found that rose bengal has a strong tendency to form a charge transfer complex with SWCNTs, and these complexes appear to be essential in the functionalization reaction.We have studied the complexation through a spectroscopic study of SWCNT fluorescence quenching and the appearance of a charge transfer absorption feature near 600 nm. Both are interpreted as results of nanotube p-doping when the dye adsorbs to the sidewall. We also observe that the charge transfer complex formation accelerates when the rose bengal is optically excited, apparently proceeding through the dye’s triplet state. A correlation between extent of guanine functionalization and the concentration of charge transfer complexes is found as ionic strength is changed. We infer that the functionalization reaction is enabled by adsorbed photosensitizers. Interestingly, the extent of SWCNT fluorescence quenching by rose bengal is found to vary significantly with (n,m) structure. This quenching effect therefore provides a simple approach to monitor structure-specific interactions between SWCNTs and coatings, including ssDNA and conventional ionic surfactants.(1) Zheng, Y.; Bachilo, S. M.; Weisman, R. B. Controlled Patterning of Carbon Nanotube Energy Levels by Covalent DNA Functionalization. ACS Nano 13, 5222 (2019). https://doi.org/10.1021/acsnano.9b03488.

Carbon Nanotube Uptake in Photosynthetic Bacteria for Near-infrared Imaging and Enhancing Bioelectricity Generation in Living Photovoltaics

The internalization of fluorescent single-walled carbon nanotubes (SWCNTs) in photosynthetic microbes can be exploited for applications ranging from energy conversion and imaging to biomolecule delivery. The internalization of these nanoparticles are largely controlled by the permeability of the surrounding microbial cell walls, which vary in surface charge density, composition, thickness, and elasticity across different species of microbes. Since these properties play a critical role in controlling internalization, the extension of SWCNT-based technologies to prokaryotes thus requires a comparative assessment of uptake across different species.Herein, we extend a technology previously developed for internalizing SWCNTs in prokaryotes, specifically unicellular Synechocystis sp. PCC 6803, to Nostoc punctiforme, a filamentous cyanobacterial strain known to differentiate into heterocysts under nitrogen starvation. Heterocysts are specialized compartments within the filament that are capable of nitrogen fixation. These compartments possess cell walls that are distinct from vegetative cells, conferring heterocysts with relatively reduced permeability and increased resistance to mechanical and osmotic stress. Using a combination of near-infrared (NIR) fluorescence, scanning electron microscopy (SEM), and Raman spectroscopy, we investigate the effects of different cell-wall architectures and SWCNT functionalizations on internalization. We specifically examine the effects of these distinct SWCNT-cell interactions on long-term cell integrity, activity, and viability. We further show that local variations in membrane structure can dictate the extent of SWCNT association and uptake in Nostoc cells. This dependency of nanoparticle internalization on cell type can be exploited for the development of novel cell-specific staining and imaging technologies.

Constructing ultra-stable photothermal plastics assisted by carbon dots with photocaged reactivity

Constructing ultra-stable photothermal plastics assisted by carbon dots with photocaged reactivity

Aggregation Reduces Subcellular Localization and Cytotoxicity of Single-Walled Carbon Nanotubes.

The non-covalent biomolecular functionalization of fluorescent single-walled carbon nanotubes (SWCNTs) has resulted in numerous in vitro and in vivo sensing and imaging applications due to many desirable optical properties. In these applications, it is generally presumed that pristine, singly dispersed SWCNTs interact with and enter live cells at the so-called nano-biointerface, for example, the cell membrane. Despite numerous fundamental studies published on this presumption, it is known that nanomaterials have the propensity to aggregate in protein-containing environments before ever contacting the nano-biointerface. Here, using DNA-functionalized SWCNTs with defined degrees of aggregation as well as near-infrared hyperspectral microscopy and toxicological assays, we show that despite equal rates of internalization, initially aggregated SWCNTs do not further accumulate within individual subcellular locations. In addition to subcellular accumulations, SWCNTs initially with a low degree of aggregation can induce significant deleterious effects in various long-term cytotoxicity and real-time proliferation assays, which are markedly different when compared to those of SWCNTs that are initially aggregated. These findings suggest the importance of the aggregation state as a critical component related to intracellular processing and toxicological response of engineered nanomaterials.

Plasmon-induced near-infrared fluorescence enhancement of single-walled carbon nanotubes

Single-walled carbon nanotubes (SWCNTs) emit near-infrared (NIR) fluorescence that is ideal for optical sensing. However, the low quantum yields diminish the sensor’s signal-to-noise ratio and limits the penetration depths for in vivo measurements. In this study, we perform a systematic investigation of the plasmonic effects of Ag and Au nanoparticles of various geometries to tune and even enhance the fluorescence intensity of single-stranded DNA-wrapped SWCNTs (ssDNA-SWCNTs). We observe a chirality-dependent NIR fluorescence enhancement that varies with both nanoparticle shape and material, with Au nanorods increasing (7, 5) and (7, 6) chirality emissions by 80% and 60% and Ag nanotriangles increasing (7, 5) and (6, 5) emissions by 200% and 240%, respectively. The chirality-dependent enhancement was modeled using finite element modeling (FEM), which confirms contributions not only from a plasmon-induced localized increase in electron density but also from the radiative recombination of dark exciton states from the resulting electromagnetic field. Finally, we demonstrate the application of these nanoparticles in enhancing the single-molecule fluorescence of individual SWCNTs imaged in a custom-built confocal setup. The plasmonically coupled sensors show four orders of magnitude greater sensitivity towards ferricyanide, a model analyte, compared to the non-coupled sensors. Plasmonic nanoparticles thus provide a tunable means of modulating SWCNT fluorescence to study fundamental transitions of otherwise forbidden states and to improve the optical sensing performance.

Single-Walled Carbon Nanotube Probes for the Characterization of Biofilm-Degrading Enzymes Demonstrated against Pseudomonas aeruginosa Extracellular Matrices.

Hydrolase co-therapies that degrade biofilm extracellular polymeric substances (EPS) allow for a better diffusion of antibiotics and more effective treatment; current methods for quantitatively measuring the enzymatic degradation of EPS are not amendable to high-throughput screening. Herein, we present biofilm EPS-functionalized single-walled carbon nanotube (SWCNT) probes for rapid screening of hydrolytic enzyme selectivity and activity on EPS. The extent of biofilm EPS degradation is quantified by monitoring the quenching of the SWCNT fluorescence. We used this platform to screen 16 hydrolases with varying bond breaking selectivity against a panel of wild-type Pseudomonas aeruginosa and mutants deficient or altered in one or more EPS. Next, we performed concentration-dependent studies of six enzymes on two common strains found in cystic fibrosis (CF) environments and, for each enzyme, extracted three first-order rate constants and their relative contributions by fitting a parallel, multi-site degradation model, with a good model fit (R2 from 0.65 to 0.97). Reaction rates (turnover rates) are dependent on the enzyme concentration and range from 6.67 × 10-11 to 2.80 × 10-3 *s-1 per mg/mL of enzymes. Lastly, we confirmed findings from this new assay using an established crystal-violet staining assay for a subset of hydrolase panels. In summary, our work shows that this modular sensor is amendable to the high-throughput screening of EPS degradation, thereby improving the rate of discovery and development of novel hydrolases.

Dendron-Polymer Hybrids as Tailorable Responsive Coronae of Single-Walled Carbon Nanotubes.

Functional composite materials that can change their spectral properties in response to external stimuli have a plethora of applications in fields ranging from sensors to biomedical imaging. One of the most promising types of materials used to design spectrally active composites are fluorescent single-walled carbon nanotubes (SWCNTs), noncovalently functionalized by synthetic amphiphilic polymers. These coated SWCNTs can exhibit modulations in their fluorescence spectra in response to interactions with target analytes. Hence, identifying new amphiphiles with interchangeable building blocks that can form individual coronae around the SWCNTs and can be tailored for a specific application is of great interest. This study presents highly modular amphiphilic polymer-dendron hybrids, composed of hydrophobic dendrons and hydrophilic polyethylene glycol (PEG) that can be synthesized with a high degree of structural freedom, for suspending SWCNTs in aqueous solution. Taking advantage of the high molecular precision of these PEG-dendrons, we show that precise differences in the chemical structure of the hydrophobic end groups of the dendrons can be used to control the interactions of the amphiphiles with the SWCNT surface. These interactions can be directly related to differences in the intrinsic near-infrared fluorescence emission of the various chiralities in a SWCNT sample. Utilizing the susceptibility of the PEG-dendrons toward enzymatic degradation, we demonstrate the ability to monitor enzymatic activity through changes in the SWCNT fluorescent signal. These findings pave the way for a rational design of functional SWCNTs, which can be used for optical sensing of enzymatic activity in the near-infrared spectral range.

Antibody-Free Rapid Detection of SARS-CoV-2 Proteins Using Corona Phase Molecular Recognition to Accelerate Development Time.

To develop better analytical approaches for future global pandemics, it is widely recognized that sensing materials are necessary that enable molecular recognition and sensor assay development on a much faster scale than currently possible. Previously developed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) point-of-care devices are based on the specific molecular recognition using subunit protein antibodies and protein receptors that selectively capture the viral proteins. However, these necessarily involve complex and lengthy development and processing times and are notoriously prone to a loss of biological activity upon sensor immobilization and device interfacing, potentially limiting their use in applications at scale. Here, we report a synthetic strategy for nanoparticle corona interfaces that enables the molecular recognition of SARS-CoV-2 proteins without any antibody and receptor design. Our nanosensor constructs consist of poly(ethylene glycol) (PEG)—phospholipid heteropolymers adsorbed onto near-infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) that recognize the nucleocapsid (N) and spike (S) protein of SARS-CoV-2 using unique three-dimensional (3D) nanosensor interfaces. This results in rapid and label-free nIR fluorescence detection. This antibody-free nanosensor shows up to 50% sensor responses within 5 min of viral protein injections with limit of detection (LOD) values of 48 fM and 350 pM for N and S proteins, respectively. Finally, we demonstrate instrumentation based on a fiber-optic platform that interfaces the advantages of antibody-free molecular recognition and biofluid compatibility in human saliva conditions.

In vivo imaging of fluorescent single-walled carbon nanotubes within C.elegans nematodes in the near-infrared window.

Caenorhabditis elegans (C. elegans) nematodes serve as a model organism for eukaryotes, especially due to their genetic similarity. Although they have many advantages like their small size and transparency, their autofluorescence in the entire visible wavelength range poses a challenge for imaging and tracking fluorescent proteins or dyes using standard fluorescence microscopy. Herein, near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) are utilized for in vivo imaging within the gastrointestinal track of C. elegans. The SWCNTs are biocompatible, and do not affect the worms’ viability nor their reproduction ability. The worms do not show any autofluorescence in the NIR range, thus enabling the spectral separation between the SWCNT NIR fluorescence and the strong autofluorescence of the worm gut granules. The worms are fed with ssDNA-SWCNT which are visualized mainly in the intestine lumen. The NIR fluorescence is used in vivo to track the contraction and relaxation in the area of the pharyngeal valve at the anterior of the terminal bulb. These biocompatible, non-photobleaching, NIR fluorescent nanoparticles can advance in vivo imaging and tracking within C. elegans and other small model organisms by overcoming the signal-to-noise challenge stemming from the wide-range visible autofluorescence.

A Spin-Coated Hydrogel Platform Enables Accurate Investigation of Immobilized Individual Single-Walled Carbon Nanotubes.

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.

Optical Nanosensors for Real-Time Feedback on Insulin Secretion by β-Cells.

Quantification of insulin is essential for diabetes research in general, and for the study of pancreatic β-cell function in particular. Herein, fluorescent single-walled carbon nanotubes (SWCNT) are used for the recognition and real-time quantification of insulin. Two approaches for rendering the SWCNT sensors for insulin are compared, using surface functionalization with either a natural insulin aptamer with known affinity to insulin, or a synthetic lipid-poly(ethylene glycol) (PEG) (C16 -PEG(2000Da)-Ceramide),bothof which show a modulation of the emitted fluorescence in response to insulin. Although the PEGylated-lipid has no prior affinity to insulin, the response of C16 -PEG(2000Da)-Ceramide-SWCNTstoinsulin is more stable and reproducible compared to the insulin aptamer-SWCNTs. The SWCNT sensors successfully detect insulin secreted by β-cells within the complex environment of the conditioned media. The insulin is quantified by comparing the SWCNTs fluorescence response to a standard calibration curve, and the results are found to be in agreement with an enzyme-linked immunosorbent assay. This novel analytical tool for real time quantification of insulin secreted by β-cells provides new opportunities for rapid assessment of β-cell function, with the ability to push forward many aspects of diabetes research.

(Invited) Quantification of NO Concentration Via Changes in Single Walled Carbon Nanotube Fluorescence

Nitric oxide (NO) is a reactive nitrogen species that is involved in both positive and negative processes in biological systems. Researchers have been unable to agree on the correlation between NO concentration and disease progression, possibly because of NO’s short half-life and the difficulty of determining concentration with current NO sensors. Single walled carbon nanotubes (SWNT), when wrapped in single-stranded (AT)15, act as a fluorescence sensor for NO. Unfortunately, the correlation between NO concentration and the SWNT’s fluorescence intensity has been difficult to determine due to the fact that the final SWNT fluorescence intensity does not directly correlate to NO concentration and an inability to immobilize the sensor without altering its properties. Through the use of a recently developed sensor platform, systematic studies have been conducted to determine the correlation between changes in SWNT fluorescence and NO concentration. The methods used to determine equations that convert SWNT fluorescence into NO concentration will be presented, providing a method to quantify extracellular NO concentration and a blueprint for the development of equations to transform changes in SWNT fluorescence into concentrations for other reactive species.

(Invited) In Vivo and Ex Vivo Biomarker Quantification Via Single-Walled Carbon Nanotube Near-Infrared Fluorescence

Optical nanosensors hold the promise of rapid, minimally-invasive longitudal monitoring of analytes within a patient or at the bedside. Such devices will allow for informed clinical intervention based on changes in disease state profiles. We are designing nanosensors based on the optical properties of single-walled carbon nanotubes (SWCNT) for use in both in vivo and ex vivo formats. SWCNT fluorescence is in the ‘tissue-transparent’ near-infrared spectrum, allowing for fluorescence measurements through biological tissue and fluids. Further, SWCNT fluorescence is photostable and responds rapidly, allowing for multiple measurements over time. We are engineering SWCNT-based sensors such that their fluorescence emission responds to multiple cancer biomarkers and inflammatory proteins. Various deployment formats include semipermeable implantable membranes, injectable hydrogel materials, and chip-based devices. Within each device, we are comparing sensor response with its physicochemical properties to optimize sensor functionality. Finally, we are deploying each sensor device in patient serum, other clinical tissues, and in live animal models of disease. For each, we are using custom-built near-infrared probe spectroscopy with rapid point-and-detect functionality. We expect these translational sensor devices to have potential as research tools or clinical diagnostics to detect disease at early stages, evaluate disease response to treatment, or monitor disease progression. These aims will allow physicians to make rapid, informed decisions on patient prognosis and treatment.

(Invited) Tailored Functionalization of Single-Walled Carbon Nanotubes for Real-Time Monitoring of Hydrolytic Enzymes Activity

Single-walled carbon nanotubes (SWCNTs) have unique optical and physical properties, and they benefit from the ease of surface functionalization and biocompatibility. Semiconducting SWCNTs fluoresce in the near-infrared (nIR) part of the spectrum, which overlaps with the transparency window of biological samples. The SWCNTs fluorescence is sensitive to the environment, and depending on the surface functionalization, subtle changes in the proximity of the nanotube can result in significant spectral modulations. Hence, SWCNTs can be utilized as optical sensors enabling real-time detection with optical signal transduction1–3. Here, we demonstrate tailored functionalization of SWCNTs, which render the nanotubes substrate for hydrolytic enzymes. We show that the chemical composition and functional groups of the SWCNTs wrapping mediate the interaction between the nanotube and the enzymes. Our results open new avenues for monitoring enzymatic activity with fluorescent nanoparticles and hold great promise for biomedical research and applications.Reference(1) Hendler-Neumark, A.; Bisker, G. Fluorescent Single-Walled Carbon Nanotubes for Protein Detection. Sensors 2019, 19 (24), 5403.(2) Bisker, G.; Bakh, N. A.; Lee, M. A.; Ahn, J.; Park, M.; O’Connell, E. B.; Iverson, N. M.; Strano, M. S. Insulin Detection Using a Corona Phase Molecular Recognition Site on Single-Walled Carbon Nanotubes. ACS Sensors 2018, 8 (2), 367–377.(3) Bisker, G.; Dong, J.; Park, H. D.; Iverson, N. M.; Ahn, J.; Nelson, J. T.; Landry, M. P.; Kruss, S.; Strano, M. S. Protein-Targeted Corona Phase Molecular Recognition. Nat. Commun. 2016, 7, 1–14.

Towards Tailored Carbon Nanotube-Based Fluorescent Sensors

Single-walled carbon nanotubes (SWCNTs) are an emerging building block for nanoscale sensors and labels because of their unique photophysical properties. Semiconducting SWCNTs fluoresce in the tissue transparency near infrared (nIR) window (840 – 1650 nm) and do not bleach. Due to their 1D nature small perturbations in their environment strongly affect their fluorescence. The major challenges in using SWCNTS for sensing is on the one side their purification and on the other side a tailored surface chemistry for molecular recognition and photophysical signal transduction.Here, we present different approaches to tailor SWCNT surface chemistry and fluorescence emission properties for the detection of biologically important metabolites. Firstly, we tuned the sensitivity of ssDNA/SWCNTs against the neurotransmitter dopamine and different plant polyphenols by changing systematically the DNA sequence. In a more rational approach, we engineered DNA strands conjugated to small peptide sequences. In order to enable a ratio-specific linkage, we determined the amount of bound DNA on the SWCNT surface, which was further observed to play an important role in maintaining colloidal stability of the modified ssDNA/SWCNT conjugates. Finally, aptamer-based approaches were used to combine stable surface modification with target selective binding. For all these approaches we demonstrated via nIR-fluorescence spectroscopy and nIR-fluorescence stand-off imaging that tailoring these nanosensors resulted in an increased specificity against their targets, ranging from small chemical communication compounds (dopamine, H2O2) to cell wall components and secreted enzymes and metabolites. Lastly, we demonstrate that these tailored SWCNT sensors can also be transferred to purified nanotube building blocks, which enables a predictable design concept for sensor properties (emission wavelength and target specificity).