Ardemis A. Boghossian’s research while affiliated with Swiss Federal Institute of Technology in Lausanne and other places

Glucose sensing is vital for managing diabetes. However, current sensors are invasive and secrete enzymatic byproducts that are inflammatory and toxic. Though DNA-wrapped single-walled carbon nanotubes (DNA-SWCNTs) emit fluorescence that is ideal for enzyme-free optical sensing, existing approaches have yet to identify a DNA sequence that can elicit a fluorescence response to glucose. We develop an approach based on clustering to design a diverse library of 90 DNA sequences to screen for a glucose response. The most responsive sequence was further improved based on favorable mutations predicted by deep learning and pattern recognition. This combination of experimental screening and supervised and unsupervised machine learning represents a generalizable approach to developing DNA-SWCNT sensors for even the most elusive analytes. KEYWORDS: near-infrared fluorescence (NIR-II), single-walled carbon nanotubes (SWNTs or SWCNTs), continuous glucose monitoring (CGM), directed evolution, clustering, deep neural network learning, pattern recognition

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.

Glucose sensing and monitoring are crucial for biological and medical applications. Compared to existing methods, real-time detection and long-term monitoring are still required. Single-walled carbon nanotubes (SWCNTs) have excellent optical properties for sensing applications, which provide the possibility for designing a new generation of glucose sensors. In this study, we describe a method for covalently conjugate glucose oxidase (GOx) on SWCNTs as an optical glucose sensor. The functional groups are introduced by a photocatalytic reaction which acts as the handle for protein loading on SWCNTs. In this sp3 defect reaction, the optical properties of SWCNTs can be maintained. With a convenient bioconjugation reaction, the GOx could be covalently linked with SWCNTs. Compared to the non-covalent immobilization conjugates, the covalent conjugate sensor exhibits a much stronger optical response toward glucose, and the stability of the biosensor also increases in harsh conditions. At the same time, we also report the changing ratio of the original E 11 and defected E 11 * peak during the bioconjugation reaction, which is also inspiring for reaction monitoring on SWCNTs. Abstract Figure

Photosynthetic bacteria are anoxygenic microorganisms with highly versatile metabolism, as they use sunlight to oxidize a broad variety of organic compounds in addition to heterotrophic and photoautotropic alternative metabolisms. Recent advances in coupling light-harvesting microorganisms with electronic components has led to a new generation of biohybrid devices based on microbial photocatalysts. These devices are limited by the poorly conductive interface between phototrophs and synthetic materials that inhibit charge transfer. Polydopamine (PDA), produced by self-assembly of dopamine, is a very versatile and bioinspired polymer with widespread applications ¹ mostly due its ability to adhere and cover surfaces of different chemical composition. The oxidative conditions employed for the formation of this dark insoluble polymer are mild and biocompatible and have inspired scientists to develop novel nanomaterials. Post-functionalization of PDA ² also enables fine tuning of properties. Furthermore, the ability of this monomer to self-assemble and polymerize in the bacterial growth medium was considered one of the requirements of the polymer to be used as coating material beside the tunable conductive properties and the flexible structure. Biocompatibility of dopamine was tested by in vivo addition in the growth media of the photosynthetic purple non sulphur Rhodobacter (R.) sphaeroides ³ in anoxygenic conditions. This study focuses on overcoming the bottleneck of biohybrid devices through the metabolically-driven encapsulation of photosynthetic cells with a bio-inspired conductive polymer. The treated cells show preserved light absorption of the photosynthetic pigments in the presence of dopamine concentrations ranging between 0.05 – 3.5 mM. The thickness and nanoparticle formation of the membrane-associated PDA matrix was further shown to vary with the dopamine concentrations in this range. Compared to uncoated cells, the encapsulated cells show up to a 20-fold enhancement in transient photocurrent measurements under mediatorless conditions. The biologically synthesized PDA can thus act as a matrix for electronically coupling the light-harvesting metabolisms of cells with conductive surfaces. ¹ Liu, Y.; Ai, K.; Lu, L. Polydopamine and its derivative materials: Synthesis and promising applications in energy, environmental, and biomedical fields. Chem. Rev. 2014, 114, 5057–5115. ² Buscemi, G., Vona, D., Ragni, R., Comparelli, R., Trotta, M., Milano, F., Farinola, G.M., Polydopamine/Ethylenediamine Nanoparticles Embedding a Photosynthetic Bacterial Reaction Center for Efficient Photocurrent Generation. Adv. Sustainable Syst. 2021, 2000303. ³ Labarile, R.; Varsalona, M.; Vona, D.; Stufano, P.; Grattieri, M.; Farinola, G. M.; Trotta, M., A novel route for anoxygenic polymerization of dopamine via purple photosynthetic bacteria metabolism. MRS Advances 2023

Optical probes enable real-time monitoring of biomarkers in vitro, in vivo, and in cells. Probes based on the fluorescence of semiconducting single-walled carbon nanotubes (SWCNTs) specifically benefit from photostable near-infrared light emissions that are minimally absorbed by biological tissue. SWCNTs are often solubilized by DNA to enable the optical detection of specific bioanalytes. Despite efforts to engineer the selectivity of the DNA sequences towards only specific analytes of interest, these DNA-wrapped sensors are prone to optical perturbations from fluctuations in pH. In this work, we explore the fluorescence effects of DNA-SWCNT in varying pH. We observe a substantial pH effect that varies with the DNA sequence and exploit this pH sensitivity for applications in cancer detection. Using xeno nucleic acids (XNAs), we further engineer optical sensors that show resilience towards fluctuations in pH, enabling the detection of biomarkers in the absence of contributions from varying pH effects.

Conventional techniques for purifying macromolecular conjugates often require complex and costly installments that are inaccessible to most laboratories. In this work, we develop a one-step micropreparative method based on a trilayered polyacrylamide gel electrophoresis (MP-PAGE) setup to purify biological samples, synthetic nanoparticles, as well as biohybrid complexes. We apply this method to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using the conventional crush-and-soak method. MP-PAGE was also able to isolate enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, which is comparable to purities achieved through a more complex two-step purification procedure involving size exclusion and immobilized metal-ion affinity chromatography. This technique was further extended to demonstrate size-dependent separation of a commercial mixture of graphene quantum dots (GQDs) into three different fractions with distinct optical properties. Finally, MP-PAGE was used to isolate DNA–EYFP and DNA–GQD bioconjugates from their reaction mixture of DNA and EYFP and GQD precursors, samples that otherwise could not be effectively purified by conventional chromatography. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.

Living photovoltaics are microbial electrochemical devices that use whole cell–electrode interactions to convert solar energy to electricity. The bottleneck in these technologies is the limited electron transfer between the microbe and the electrode surface. This study focuses on enhancing this transfer by engineering a polydopamine (PDA) coating on the outer membrane of the photosynthetic microbe Synechocystis sp. PCC6803. This coating provides a conductive nanoparticle shell to increase electrode adhesion and improve microbial charge extraction. A combination of scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV–Vis absorption, and Raman spectroscopy measurements were used to characterize the nanoparticle shell under various synthesis conditions. The cell viability and activity were further assessed through oxygen evolution, growth curve, and confocal fluorescence microscopy measurements. The results show sustained cell growth and detectable PDA surface coverage under slightly alkaline conditions (pH 7.5) and at low initial dopamine (DA) concentrations (1 mM). The exoelectrogenicity of the cells prepared under these conditions was also characterized through cyclic voltammetry (CV) and chronoamperometry (CA). The measurements show a three-fold enhancement in the photocurrent at an applied bias of 0.3 V (vs. Ag/AgCl [3 M KCl]) compared to non-coated cells. This study thus lays the framework for engineering the next generation of living photovoltaics with improved performances using biosynthetic electrodes.

Recent advances in coupling light-harvesting microorganisms with electronic components have led to a new generation of biohybrid devices based on microbial photocatalysts. These devices are limited by the poorly conductive interface between phototrophs and synthetic materials that inhibit charge transfer. This study focuses on overcoming this bottleneck through the metabolically-driven encapsulation of photosynthetic cells with a bio-inspired conductive polymer. Cells of the purple non sulfur bacterium Rhodobacter sphaeroides were coated with a polydopamine (PDA) nanoparticle layer via the self-polymerization of dopamine under anaerobic conditions. The treated cells show preserved light absorption of the photosynthetic pigments in the presence of dopamine concentrations ranging between 0.05–3.5 mM. The thickness and nanoparticle formation of the membrane-associated PDA matrix were further shown to vary with the dopamine concentrations in this range. Compared to uncoated cells, the encapsulated cells show up to a 20-fold enhancement in transient photocurrent measurements under mediatorless conditions. The biologically synthesized PDA can thus act as a matrix for electronically coupling the light-harvesting metabolisms of cells with conductive surfaces.

Osteoarthritis (OA) is a degenerative joint disease affecting millions worldwide. Early detection of OA and monitoring its progression is essential for effective treatment and for preventing irreversible damage. Although sensors have emerged as a promising tool for monitoring analytes in patients, their application for monitoring the state of pathology is currently restricted to specific fields (such as diabetes). In this study, we present the development of an optical sensor system for real-time monitoring of inflammation based on the measurement of nitric oxide (NO), a molecule highly produced in tissues during inflammation. Single-walled carbon nanotubes (SWCNT) were functionalized with a single-stranded DNA (ssDNA) wrapping designed using an artificial intelligence approach and tested using S-nitroso-N-acetyl penicillamine (SNAP) as a standard released-NO marker. An optical SWIR reader with LED excitation at 650 nm, 730 nm and detecting emission above 1000 nm was developed to read the fluorescence signal from the SWCNTs. Finally, the SWCNT was embedded in GelMa to prove the feasibility of monitoring the release of NO in bovine chondrocyte and osteochondral inflamed cultures (1–10 ng/ml IL1β) monitored over 48 hours. The stability of the inflammation model and NO release was indirectly validated using the Griess and DAF-FM methods. A microfabricated sensor tag was developed to explore the possibility of using ssDNA-SWCNT in an ex vivo anatomic set-up for surgical feasibility, the limit of detection, and the stability under dynamic flexion. The SWCNT sensor was sensitive to NO in both in silico and in vitro conditions during the inflammatory response from chondrocyte and osteochondral plug cultures. The fluorescence signal decreased in the inflamed group compared to control, indicating increased NO concentration. The micro-tag was suitable and stable in joints showing a readable signal at a depth of up to 6 mm under the skin. The ssDNA-SWCNT technology showed the possibility of monitoring inflammation continuously in an in vitro set-up and good stability inside the joint. However, further studies in vivo are needed to prove the possibility of monitoring disease progression and treatment efficacy in vivo . Acknowledgments : The project was co-financed by Innosuisse (grant nr. 56034.1 IP-LS)

Single-walled carbon nanotubes (SWCNTs) show great potential for biosensing applications due to their unique optical properties. SWCNTs emit fluorescence in the near-infrared (NIR) region that is stable, biotransparent, and highly sensitive to changes in their environment. The biocompatibility and the selectivity of their interactions can be engineered through SWCNT surface functionalization. One of the most common functionalization approaches is the non-covalent wrapping of SWCNTs with single-stranded DNA (ssDNA). Though the ssDNA sequences can be varied to control the optical response of these SWCNT sensors, the dependence of the optical response on the sequence is unknown and unpredictable. This lack of information on the relationship between the ssDNA sequence and sensor performance is a major bottleneck in engineering SWCNT-based optical sensors. In this work, we develop a guided approach to engineer optical ssDNA-SWCNT sensors. Using a combination of machine learning and directed evolution [1] , we designed an optical sensor for nitric oxide (NO), a pro-inflammatory mediator that induces inflammation [2] . We demonstrate a 7-fold enhancement in sensor response compared to the state-of-the-art sensor based on the (AT) 15 sequence [3] . This approach thus provides a powerful means of overcoming existing bottlenecks in SWCNT sensor design, ushering a new generation of near-infrared technologies for biomedical research and clinical diagnostics. References [1] B. Lambert, A. J. Gillen, N. Schuergers, S. J. Wu, A. A. Boghossian, Chem. Commun. 2019 , 55 , 3239. [2] J. N. Sharma, A. Al-Omran, S. S. Parvathy, Inflammopharmacology 2007 , 15 , 252. [3] J. Zhang, A. A. Boghossian, P. W. Barone, A. Rwei, J. H. Kim, D. Lin, D. A. Heller, A. J. Hilmer, N. Nair, N. F. Reuel, M. S. Strano, J. Am. Chem. Soc. 2011 , 133 , 567.