Alessandra and Shang-Jung present posters at NT19

Spinning-Disc Confocal Microscopy in the Second Near-Infrared Window (NIR-II)

Alessandra Antonucci, Nils Schuergers, Benjamin Lambert, Andrea Latini, Raino Ceccarelli, Andrea Santinelli, Andrii Rogov, Daniel Ciepielewski, Ardemis 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 autofluores‐ cence within this range, offers increased resolution and contrast with even greater pene‐ tration 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% enhance‐ ment 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.

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

Alessandra Antonucci, Nils Schuergers, Vitalijs Zubkovs, Ardemis Boghossian

The distinctive properties of single‐walled carbon nanotubes (SWCNTs) have inspired many innovative applications in the field of cell nanobiotechnology. Most studies to date have focused on eukaryotic cells capable of internalizing functionalized SWCNTs, however, the effect of SWCNT functionalization on transport across the thick cell wall of prokaryotes re‐ mains unexplored. Herein, we explore SWCNT uptake in Gram‐negative cyanobacteria and demonstrate selective internalization of SWCNTs decorated with charged protein wrappings. These functionalized SWCNTs are shown to traverse the outer cell wall of filamentous and unicellular strains of cyanobacteria, independent natural competency for DNA uptake, with adsorption and internalization rate constants of kads = (9.08 ± 0.16)x10−8 s−1 and kin = (1.466 ± 0.011)x10−4 s−1, respectively. A custom‐built, spinning disc confocal microscope enabled direct imaging of the near‐infrared (NIR) SWCNT fluorescence within cells, revealing a highly inhomogeneous distribution of SWCNTs, that is 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.

Directed Evolution of the Optoelectronic Properties of Synthetic Nanomaterials

Shang-Jung Wu, Benjamin Lambert, Alice Judith Gillen, Nils Schuergers, Ardemis Boghossian

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.

The Impact of Exposed Surface Area on the Response of SWCNT Optical Sensors

Shang‐Jung Wu, Alice Judith Gillen, Daniel Jerome Siefman, Claire Bourmaud, Benjamin Lambert, Ardemis Boghossian

Due to their distinct and advantageous fluorescence properties, semiconducting single‐ walled carbon nanotubes (SWCNTs) are being applied to a variety of optical sensing appli‐ cations. 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 fo‐ cus 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 mecha‐ nisms governing the selectivity of existing SWCNT‐based optical sensors.

Mediatorless, Reversible Optical Nanosensor Enabled through Enzymatic Pocket Doping

Alessandra Antonucci, Vitalijs Zubkovs, Nils Schuergers, Benjamin Lambert, Esra Ahunbay, Ardemis Boghossian

The existing single‐walled carbon nanotube (SWCNT) optical sensors that rely on charge transfer for signal transduction often require exogenous mediators that compromise the sta‐ bility 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 mem‐ brane device, the sensor demonstrates a monotonic increase in initial response rates with in‐ creasing glucose concentrations between 3×10−3 and 30x10−3M and an apparent Michaelis– Menten constant of KM (app)≈ 13.9x10−3 M. A combination of fluorescence, absorption, and Raman spectroscopy measurements suggests a fluorescence enhancement mecha‐ nism 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 recov‐ ery 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.

Xeno Nucleic Acid Nanosensors for Enhanced Stability

Shang‐Jung Wu, Alice Judith Gillen, Justyna Kupis‐Rozmysłowicz, Carlo Gigli, Nils Schuergers, Ardemis 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 af‐ fect 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 concen‐ trations 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 nan‐ otube optoelectronic behaviour, unlocking previously unexplored possibilities for developing nano‐bioengineered sensors with augmented capabilities.

Characterization of Double-Stranded DNA on Single-Walled Carbon Nanotubes (SWCNTs)

Shang‐Jung Wu, Nils Schuergers, Kun‐Han Lin, Alice Judith Gillen, Clémence Corminboeuf, Ardemis Boghossian

DNA has been extensively studied due to its versatility as a dispersant of SWCNTs. Its speci‐ ficity and binding affinity to various analytes has exhibited a strong sequence dependence. 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 vivo imaging, and chirality separation. To date, most research has focused on the interaction between single‐stranded DNA and SWCNT surface, with less fo‐ cus on the nature of the interaction of double‐stranded DNA (dsDNA) and SWCNTs. As a result, the exact interaction mechanism governing this type of complex remains strongly de‐ bated 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.