Covalent quantum color centers, (CCs) introduced onto single-walled carbon nanotubes (SWCNTs) create localized, red-shifted emission states with promising applications in quantum photonics, biosensing and bioimaging. Yet, the mechanisms governing where and how these color centers form on the SWCNTs remain poorly understood. To address this question, we mapped, in real time, the precise position of aryl CCs formed by photoinduced diazonium chemistry along individual (6,5) SWCNTs by combining, within a correlative framework, two complementary super-resolution imaging modalities: single-molecule localization microscopy of individual CC formation events and analytical reconstruction of the SWCNT backbone. We reveal that CC formation is not spatially random as CCs preferentially localize towards the central regions of nanotubes, away from their ends, and exhibit pronounced spatial clustering. Ruling out curvaturedriven effects, we attribute this clustering to non-uniform surfactant coverage along the nanotube, which creates locally exposed and chemically-reactive domains. These results establish nanotube surface heterogeneity as a key determinant of CC positioning and point toward strategies for the deterministic, spatially controlled engineering of quantum emitters on carbon nanotubes.

Single-walled carbon nanotubes (SWCNTs) are wrapped with single-stranded DNA (ssDNA) to create near-infrared (NIR-II) fluorescent sensors for diverse analytes. However, the interaction between the negatively charged backbone of ssDNA and cations in biological saline alters fluorescence unpredictably. This susceptibility limits the application of these sensors in biological media. To address this limitation, this study develops a cation-pretreatment strategy that quenches the baseline fluorescence of ssDNA-SWCNTs to enable turn-on responses that are selectively triggered by analytes in saline. An initial screening of Na+, K+, Mg2+, Ca2+, and Al3+ pretreatments of gel-encapsulated (AT)15-SWCNTs reveals that Al3+ pretreatment induces a stable quenching of fluorescence that is reversible only on Al3+ chelation or precipitation. We apply this Al3+ pretreatment to develop a saline-resilient, near-infrared sensor for dopamine. The Al3+-treated (AT)15-SWCNTs show a concentration- and chirality-dependent fluorescence response over a dynamic range of 1 nM and 10 μM dopamine, achieving a 110-fold increase in the turn-on response to 10 mM dopamine in buffered saline compared with the untreated (AT)15-SWCNTs. Further study of the effects of pH and different salts on the dopamine response suggests a mechanism that relies on competing trivalent cations and negative DNA phosphate interactions. These interactions lay the framework for saline-resilient optical sensors that exploit DNA as a charged-based actuator for modulating the exciton dynamics and controlling the SWCNT fluorescence.

A strategy is presented for the asymmetric chemical functionalization of individual single-walled carbon nanotubes (SWCNTs) termini and their selective conjugation to distinct single moieties, i.e., the formation of bi-functionalized R-CNT-R' heterostructures. This study demonstrates this via the selective covalent attachment of metal nanoparticles (NPs) and semiconductor nanocrystals (quantum dots, QDs) at opposite ends of individual SWCNTs; the electronic coupling is further characterized between the distinct components of the hybrids. The general applicability of the methodology developed by this study can be beneficial for the design and development of novel SWCNT-based nanohybrid materials with single-molecule control.

During liver fibrosis, recurrent hepatic injuries lead to the accumulation of collagen and other extracellular matrix components in the interstitial space, ultimately disrupting liver functions. Early stages of liver fibrosis may be reversible, but opportunities for diagnosis at these stages are currently limited. Here, we show that the alterations of the interstitial space associated with fibrosis can be probed by tracking individual fluorescent single-walled carbon nanotubes (SWCNTs) diffusing in that space. In a mouse model of early liver fibrosis, we find that nanotubes generally explore elongated areas, whose lengths decrease as the disease progresses, even in regions where histopathological examination does not reveal fibrosis yet. Furthermore, this decrease in nanotube mobility is a purely geometrical effect as the instantaneous nanotube diffusivity stays unmodified. This work establishes the promise of SWCNTs both for diagnosing liver fibrosis at an early stage and for more in-depth studies of the biophysical effects of the disease.

DNA-wrapped single-walled carbon nanotubes (SWCNTs) have demonstrated great versatility in their use as optical sensors. SWCNTs emit a near-infrared fluorescence that is responsive to even the slightest changes in the nanotube environment, enabling sensors that can respond to single-molecule fluctuations within the vicinity of their surfaces. The fluorescence response and surface interactions of these sensors are determined by the DNA wrapping sequence. However, the lack of information on the relationship between the DNA sequence and its effect on the SWCNT fluorescence remains a bottleneck for designing sequences that are specific to analytes of interest. We have recently demonstrated the use of directed evolution to control the fluorescence response of SWCNTs through DNA design. Iterative cycles of DNA mutation, screening, and selection allowed us to evolve sequences that yield DNA-wrapped SWCNT sensors with a desired fluorescence response to mycotoxins. In this work, we apply the screening results of the DNA libraries used in this approach to train machine learning (ML) algorithms. Artificial neural network (ANN) and support vector machine (SVM) methods were used to predict the response of ssDNA-SWCNT sensors to a specific mycotoxin. The reliability of these models was further assessed through cross-validation. The ANN and SVM models with cross-validation were able to accurately classify the various DNA sequences as yielding either a high or low fluorescence response with an accuracy of 73 and 81%, respectively. The models were further used to predict the performance of alternative DNA sequences outside the initial training dataset, using the Hierarchy and k-means ++ clustering methods to evaluate the similarity and dissimilarity of each DNA sequence. Compared to the SVM model, the ANN model showed an improved ability to predict high responses for dissimilar DNA sequences. We further applied a combinatorial approach based on SVM and ANN models to design new DNA sequences for improving sensor performance. The success of this approach was validated experimentally, demonstrating the rational design of improved sensors with 95% prediction accuracy. The application of ML algorithms to directed evolution libraries of DNA thus allows one to accurately map the performances of these sensors within a particular sequence space. The computational success of this mapping provides a framework for replacing current empirical approaches with the rational design of DNA sequences for SWCNT sensing. Keywords: Nano Biosensor, DNA-SWCNT, Mycotoxin, Machine learning, Directed evolution method

In this study, we develop and apply a directed evolution approach to engineer the optical sensing properties of DNA-wrapped single-walled carbon nanotubes (DNA-SWCNTs) towards mycotoxins, a class of molecules critical to detect in the food industry. We successfully demonstrate the creation of sensors for the detection of both the aflatoxin B1 (AFB1) and fumonisin B1 (FB1) mycotoxins based on the specific response of the (9,4) and (7,5) SWCNT chirality fluorescence peaks, respectively. The resulting chirality-specific responsivity was used to demonstrate the multimodal detection of both mycotoxins at different wavelengths of light in the presence of complex food medium. Moreover, we show that directed evolution can be used not only to improve the chiral-dependent selectivity of our sensors to the mycotoxins, but also the sensor sensitivity and fluorescence intensity through multiple rounds of evolution. The approach demonstrated in this study is versatile and could be generalized to other SWCNT sensors as well as other nanosensors comprising a biological element.

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.

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.

Properties of SWCNT-based sensors such as brightness and detection capabilities strongly depend on the characteristics of the wrapping used to suspend the nanotubes. In this study, we explore ways to modify the properties of DNA-SWCNT sensors by using chemically modified DNA sequences, with the aim of creating sensors more suitable for use in in vivo and in vitro applications. We show that both the fluorescence intensity and sensor reactivity are strongly impacted not only by the chemical modification of the DNA but also by the method of preparation. In the absence of modifications, the sensors prepared using MeOH-assisted surfactant exchange exhibited higher overall fluorescence compared to those prepared by direct sonication. However, we demonstrate that the incorporation of chemical modifications in the DNA sequence could be used to enhance the fluorescence intensity of sonicated samples. We attribute these improvements to both a change in dispersion efficiency as well as to a change in SWCNT chirality distribution. Furthermore, despite their higher intensities, the response capabilities of sensors prepared by MeOH-assisted surfactant exchange were shown to be significantly reduced compared to their sonicated counterparts. Sonicated sensors exhibited a globally higher turn-on response towards dopamine compared to the exchanged samples, with modified samples retaining their relative intensity enhancement. As the increases in fluorescence intensity were achieved without needing to alter the base sequence of the DNA wrapping or to add any exogenous compounds, these modifications can - in theory - be applied to nearly any DNA sequence to increase the brightness and penetration depths of a variety of DNA-SWCNT sensors without affecting biocompatibility or reducing the near-limitless sequence space available. This makes these sensors an attractive alternative for dopamine sensing in vitro and in vivo by enabling significantly higher penetration depths and shorter laser exposure times.

Bioengineers have mastered practical techniques for tuning a biomaterial's properties with only limited information on the relationship between the material's structure and function. These techniques have been quintessential to engineering proteins, which are most often riddled with ill-defined structure-function relationships. In this Perspective, we review bioengineering approaches aimed at overcoming the elusive protein structure-function relation. We extend these principles to engineering synthetic nanomaterials, specifically applying the underlying theory to optical sensors based on single-stranded DNA-wrapped single-walled carbon nanotubes (ssDNA-SWCNTs). Bioengineering techniques such as directed evolution, computational design, and noncanonical synthesis are reviewed in the broader context of nanomaterials engineering. We further provide an order-of-magnitude analysis of empirical approaches that rely on random or guided searches for designing new nanomaterials. The underlying concepts presented in these approaches can be further extended to a broad range of engineering fields confronted with empirical design strategies, including catalysis, metal-organic frameworks (MOFs), pharmaceutical dosing, and optimization algorithms.

This experimental study investigates a novel multi-functional one dimensional-two dimensional (1D-2D) heterojunction made of two different band-gap semiconductors, i.e. single-walled carbon nanotube (SWCNT) and tungsten di-selenide (WSe 2 ). The proposed ultra-scaled van der Waals junction behaves like a p-type field-effect transistor (FET) or a gated p-n diode, depending upon the gate bias. A detailed physical study of charge transportation mechanism is presented in a wide range of applied potentials. The obtained results are important towards scaling nanoelectronics to atomic thicknesses by means of the 1D-2D hybrid junctions with additional multi-functional solid-state devices.

Directed evolution is a powerful approach for tailoring protein properties toward new or enhanced functions. This technique allows one to alter a protein in a directed manner even in the absence of a known relationship between protein structure and function. Several nanocomplexes, such as optical sensors based on DNA-wrapped single-walled carbon nanotubes (DNA-SWCNTs), similarly lack a known structure-function relationship. For example, DNA-SWCNT complexes exhibit variations in their fluorescence that depend on the sequence of the DNA wrapping. However, little is known about the relationship between the DNA sequence and its effects on the fluorescence properties of SWCNTs. In this study, we use directed evolution as a guided approach to tuning the optoelectronic properties of DNA-SWCNT complexes through DNA mutation. Applying this technique over two rounds of DNA mutagenesis and screening, we evolved a DNA-SWCNT sensor with a 57% enhancement in fluorescence intensity. Furthermore, this sensor shows retained selectivity and sensitivity towards the analyte of interest. Though we demonstrate this approach to improve sensor brightness, directed evolution offers far-reaching possibilities in the field that can also be applied to improve additional sensor properties such as sensitivity and selectivity.

Directed evolution is a powerful approach to tailor protein properties toward new or enhanced functions. Herein, we use directed evolution to engineer the optoelectronic properties of DNA-wrapped single-walled carbon nanotube sensors through DNA mutation. This approach leads to an improvement in the fluorescence intensity of 56% following two evolution cycles.

Surfactants offer a tunable approach for modulating the exposed surface area of a nanoparticle. They further present a scalable and cost-effective means for suspending single-walled carbon nanotubes (SWCNTs), which have demonstrated practical use as fluorescence sensors. Though surfactant suspensions show record quantum yields for SWCNTs in aqueous solutions, they lack the selectivity that is vital for optical sensing. We present a new method for controlling the selectivity of optical SWCNT sensors through colloidal templating of the exposed surface area. Colloidal nanotube sensors were obtained using various concentrations of sodium cholate, and their performances were compared to DNA-SWCNT optical sensors. Sensor responses were measured against a library of bioanalytes, including neurotransmitters, amino acids, and sugars. We report an intensity response towards dopamine and serotonin for all sodium cholate-suspended SWCNT concentrations. We further identify a selective, 14.1 nm and 10.3 nm wavelength red-shifting response to serotonin for SWCNTs suspended in 1.5 and 0.5 mM sodium cholate, respectively. Through controlled, adsorption-based tuning of the nanotube surface, this study demonstrates the applicability of sub-critical colloidal suspensions to achieve selectivities exceeding those previously reported for DNA-SWCNT sensors.

Due to their distinct and advantageous fluorescence properties, semiconducting single-walled carbon nanotubes (SWCNTs) are being applied to a variety of optical sensing applications. Limitations in solubility and biocompatibility have been overcome by non-covalently functionalizing the surface of the SWCNT with wrappings using techniques that retain its inherent fluorescence. Though wrappings based on surfactants and single-stranded DNA (ssDNA) have been extensively studied for this purpose, they are limited by factors such as lack of selectivity and lower fluorescence quantum yield, respectively. In this study, we take advantage of the higher fluorescence emission of surfactant-coated SWCNTs and focus on new approaches that can tune their selectivity as sensors. Through the concomitant monitoring of both the fluorescence intensity and wavelength position of emission peaks, we demonstrate the ability to increase the selectivity of sensors based on surfactant-suspended SWCNTs while retaining their higher fluorescence intensity. These results provide not only a promising avenue for rationally designing SWCNT sensors, but also insight on the mechanisms governing the selectivity of existing SWCNT-based optical sensors.

Fluorescence microscopy in the second near-infrared optical window (NIR-II, 1000–1350 nm) has become a technique of choice for non-invasive in vivo imaging. The deep penetration of NIR light in living tissue, as well as negligible tissue autofluorescence within this optical range, offers increased resolution and contrast with even greater penetration depths. Here, we present a custom-built spinning-disc confocal laser microscope (SDCLM) that is specific to imaging in the NIR-II. The SDCLM achieves a lateral resolution of 0.5 ± 0.1 µm and an axial resolution of 0.6 ± 0.1 µm, showing a ~17% and ~45% enhancement in lateral and axial resolution, respectively, compared to the corresponding wide-field configuration. We furthermore showcase several applications that demonstrate the use of the SDCLM for in situ , spatiotemporal tracking of NIR particles and bioanalytes within both synthetic and biological systems.

The sensitivity of fluorescent SWCNT-based sensors has been shown to strongly depend on sensor brightness, with the brightest sensors demonstrating enhanced sensitivity capable of single-molecule detection. Although defect-free nanotubes show improved brightness that can enhance sensor sensitivity, the applicability of these nanotubes are often limited by their availability and the sensor preparation procedure, which can introduce defects that diminish quantum yield. Sensor brightness can be tuned to a certain degree through the non-covalent functionalization of SWCNTs with various single-stranded DNA (ssDNA) sequences. However, the correlation between sequence identity and SWCNT brightness remains elusive, and an empirical approach must thus be used to improve brightness. Here, we present a systematic approach to modulating SWCNT brightness through chemical modification of DNA. These modifications can be applied to nearly any DNA sequence, allowing one to enhance SWCNT brightness without significantly altering the sequence-based specificity conferred by the DNA wrapping.

Single-walled carbon nanotubes (SWCNTs) exhibit intrinsic near-infrared fluorescence that benefits from indefinite photostability and tissue transparency, offering a promising basis for in vivo biosensing. Existing SWCNT optical sensors that rely on charge transfer for signal transduction often require exogenous mediators that compromise the stability and biocompatibility of the sensors. This study presents a reversible, mediatorless, near-infrared glucose sensor based on glucose oxidase-wrapped SWCNTs (GOx-SWCNTs). GOx-SWCNTs undergo a selective fluorescence increase in the presence of aldohexoses, with the strongest response toward glucose. When incorporated into a custom-built membrane device, the sensor demonstrates a monotonic increase in initial response rates with increasing glucose concentrations between 3 × 10-3 and 30 × 10-3 m and an apparent Michaelis-Menten constant of KM (app) ≈ 13.9 × 10-3 m. A combination of fluorescence, absorption, and Raman spectroscopy measurements suggests a fluorescence enhancement mechanism based on localized enzymatic doping of SWCNT defect sites that does not rely on added mediators. Removal of glucose reverses the doping effects, resulting in full recovery of the fluorescence intensity. The cyclic addition and removal of glucose is shown to successively enhance and recover fluorescence, demonstrating reversibility that serves as a prerequisite for continuous glucose monitoring.