The LNB presents posters at SCS Fall Meeting

Xeno Nucleic Acid Nanosensors for Enhanced Stability

Alice J. Gillen, Justyna Kupis-Rozmysłowicz, Carlo Gigli, Nils Schuergers, Ardemis A. Boghossian

The omnipresence of salts in biofluids creates a pervasive challenge in designing sensors suitable for in vivo applications. Fluctuations in ion concentrations have been shown to affect the sensitivity and selectivity of optical sensors based on single-walled carbon nanotubes wrapped with single-stranded DNA (ssDNA-SWCNTs). We herein observe fluorescence wavelength shifting for ssDNA-SWCNT-based optical sensors in the presence of divalent cations at concentrations above 3.5 mM. In contrast, no shifting was observed for concentrations up to 350 mM for sensors bioengineered with increased rigidity using xeno nucleic acids (XNAs). Transient fluorescence measurements reveal distinct optical transitions for ssDNA- and XNA-based wrappings during ion-induced conformation changes, with XNA-based sensors showing increased permanence in conformational and signal stability. This demonstration introduces synthetic biology as a complementary means for enhancing nanotube optoelectronic behaviour, unlocking previously unexplored possibilities for developing nano-bioengineered sensors with augmented capabilities.

Restriction Enzyme Analysis of Double-Stranded DNA on Pristine Single-Walled Carbon Nanotubes

Shang-Jung Wu, Nils Schuergers, Kun-Han Lin, Alice J. Gillen, Clémence Corminboeuf, Ardemis A. Boghossian

DNA has been extensively studied due to its versatility as a dispersant of SWCNTs. The specificity and binding affinity of DNA to various analytes has been shown to be strongly sequence dependent. As a result, through modifications of the nucleotide sequence, the DNA conformation on the SWCNT surface can be tailored to suit specific needs for techniques such as single-molecule detection, in vivoimaging, and chirality separation. To date, the majority of research has focused on the interaction between single-stranded DNA and SWCNT surface, with less focus on the nature of the interaction between double-stranded DNA (dsDNA) and SWCNTs. As a result, the exact interaction mechanism governing this type of complex remains strongly debated and largely unknown. In this study, we employ various biochemical methods to infer the conformation of dsDNA on the surface of SWCNTs. Our methods are based on imaging techniques for identifying DNA-modifying enzyme activity in the presence of dsDNA-SWCNT complexes. Our findings suggest that dsDNA can partially retain its native conformation on the SWCNT surface, and the degree of dsDNA accessibility is strongly sequence dependent. These findings offer new possibilities for SWCNT sensing applications that employ dsDNA, such as the optical detection of DNA-protein interactions

Near-Infrared Confocal Imaging of Single-Walled Carbon Nanotube Uptake in Bacteria

Alessandra Antonucci, Nils Schuergers, Vitalijs Zubkovs, Ardemis A. Boghossian

The distinctive properties of single-walled carbon nanotubes (SWCNTs) have inspired the development of innovative applications in the field of cell nanobiotechnology. Most studies to date have focused on eukaryotic cells capable of internalizing SWCNTs functionalized with a variety of non-covalent wrappings. However, the effect of SWCNT functionalization on transport across the thick cell wall of prokaryotes remains unexplored. In this study, we explore the uptake of SWCNTs in Gram-negative cyanobacteria and demonstrate selective internalization of SWCNTs decorated with charged protein wrappings. The functionalized SWCNTs are shown to traverse the outer cell wall of both filamentous and unicellular strains of cyanobacteria, independent of whether the strain is naturally competent for DNA uptake, with adsorption and internalization rate constants of k_ads = (9.08 ± 0.16) x 10^-8s^-1 and k_in = (1.466 ± 0.011) x 10^-4s^-1, respectively. A custom-built, spinning disc confocal microscope was used for the first time to directly image near-infrared (NIR) SWCNT fluorescence within cells, revealing a highly inhomogeneous distribution of SWCNTs that is otherwise overlooked using conventional NIR widefield imaging. The nanobionic cells show sustained photosynthetic activity and growth, offering a powerful avenue for engineering photosynthetic organisms with augmented and even inherited nanobionic capabilities.

Spinning-disc confocal microscopy in the second near-infrared window

Vitalijs Zubkovs, Alessandra Antonucci, Nils Schuergers, Benjamin Lambert, Andrea Latini, Raino Ceccarelli, Andrea Santinelli, Andrii Rogov, Daniel Ciepielewski, Ardemis A. Boghossian

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 minimal or complete absence of tissue autofluorescence within this 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 resolutions, 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.

Mediatorless, Reversible Optical Nanosensor Enabled through Enzymatic Pocket Doping

Vitalijs Zubkovs, Nils Schuergers, Benjamin Lambert, Esra Ahunbay, Ardemis A. Boghossian

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 × 10⁻³and 30 × 10⁻³ M and an apparent Michaelis–Menten constant of K_M(app) ≈ 13.9 × 10⁻³M. 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.