Detection and Imaging of the Plant Pathogen Response by Near‐Infrared Fluorescent Polyphenol Sensors

Plants use secondary metabolites such as polyphenols for chemical defense against pathogens and herbivores. Despite their importance in plant pathogen interactions and tolerance to diseases, it remains challenging to detect polyphenols in complex plant tissues. Here, we create molecular sensors for plant polyphenol imaging that are based on near-infrared (NIR) fluorescent single-wall carbon nanotubes (SWCNTs). We identified polyethylene glycol–phospholipids that render (6,5)-SWCNTs sensitive (K$_{d}$=90 nM) to plant polyphenols (tannins, flavonoids, …), which red-shift (up to 20 nm) and quench their emission (ca. 1000 nm). These sensors report changes in total polyphenol level after herbivore or pathogen challenge in crop plant systems (Soybean Glycine max) and leaf tissue extracts (Tococa spp.). We furthermore demonstrate remote chemical imaging of pathogen-induced polyphenol release from roots of soybean seedlings over the time course of 24 h. This approach allows in situ visualization and understanding of the chemical plant defense in real time and paves the way for plant phenotyping for optimized polyphenol secretion

Protein Aggregation on Metal Oxides Governs Catalytic Activity and Cellular Uptake.

Engineering of catalytically active inorganic nanomaterials holds promising prospects for biomedicine. Catalytically active metal oxides show applications in enhancing wound healing but have also been employed to induce cell death in photodynamic or radiation therapy. Upon introduction into a biological system, nanomaterials are exposed to complex fluids, causing interaction and adsorption of ions and proteins. While protein corona formation on nanomaterials is acknowledged, its modulation of nanomaterial catalytic efficacy is less understood. In this study, proteomic analyses and nano-analytic methodologies quantify and characterize adsorbed proteins, correlating this protein layer with metal oxide catalytic activity in vitro and in vivo. The protein corona comprises up to 280 different proteins, constituting up to 38% by weight. Enhanced complement factors and other opsonins on nanocatalyst surfaces lead to their uptake into macrophages when applied topically, localizing >99% of the nanomaterials in tissue-resident macrophages. Initially, the formation of the protein corona significantly reduces the nanocatalysts' activity, but this activity can be partially recovered in endosomal conditions due to the proteolytic degradation of the corona. Overall, the research reveals the complex relationship between physisorbed proteins and the catalytic characteristics of specific metal oxide nanoparticles, providing design parameters for optimizing nanocatalysts in complex biological environments

Tailored near-infrared fluorescent carbon nanotube sensors for pathogen detection

Nutzen und Notwenigkeit schneller und spezifischer Analysemethoden wurden spätestens während der globalen COVID-19 Pandemie jedem deutlich. Vor allem Entwicklungen im Bereich der Nanotechnologie könnten zu bislang ungeahnten medizinischen Diagnostikmethoden führen. Bislang sind jedoch kaum optische Nanosensoren zur Pathogendetektion bekannt, könnten aber in Zukunft eine schnelle, markierungsfreie und ortsaufgelöste Identifikation bakterieller Infektionen ermöglichen. Eine Klasse an Nanomaterialien mit außergewöhnlichen photophysikalischen Eigenschaften, welche sich zum Aufbau solcher speziellen Biosensoren eignet, sind halbleitende, einwandige Kohlenstoff-Nanoröhren (SWCNTs). Diese röhrenförmigen Kohlenstoffallotrope besitzen eine Durchmesser-, genauer „Chiralitäts“-abhängige Bandlücke. Dies ermöglicht eine Fluoreszenzemission (900-1700 nm) im nah-Infrarot (NIR), ein spektraler Bereich, besonders geeignet für die Sensorik in biologischen Systemen. Bevor jedoch diese Nanopartikel in der Diagnostik eingesetzt werden können, muss deren hydrophobe Oberfläche nicht-kovalent modifiziert werden, was maßgeblich die spätere Funktionalität und kolloidale Stabilität beeinflusst. Diese funktionalisierten SWCNTs sind in der Lage Änderungen der lokalen chemischen Umgebung in Fluoreszenzsignale zu übersetzen, was das Grundprinzip dieser optischen Sensoren darstellt. Die vorliegende Arbeit beschäftigt sich mit der zielgerichteten Optimierung der Oberflächenchemie und optischen Eigenschaften der SWCNT-Sensoren, mit dem Ziel, Pathogene und Pathogeninteraktionen zu detektieren. Hierzu wurde 1) eine Methode entwickelt, um die Oberflächenmodifikation der SWNCTs im Detail zu verstehen, genauer, um die Menge adsorbierter einzelsträngiger DNA-Polymere zu quantifizieren. Die Resultate zeigten auf, dass auf einer Nanoröhre mehrere hundert DNA-Moleküle adsorbieren, abhängig von der Länge und Zusammensetzung der Oligonukleotide. Aufgrund dieser Quantifizierung konnten verhältnis-spezifische bioorthogonale Modifikationen der Nanokomplexe durchgeführt werden. 2) Zur Optimierung der Nanosensoren wurden einzelne SWCNT-Chiralitäten isoliert, welche definierte Emissionsspektren besitzen. Dazu wurde zuerst die Fähigkeit bestimmter Polyfluorenpolymere ausgenutzt, selektiv SWCNTs zu dispergieren, sodass diese nach Austausch der Oberflächenmodifikation in biologischen Systemen eingesetzt werden können. Ein weiterer Ansatz separierte SWCNTs mit Hilfe der wässrigen Zweiphasenextraktion und führte nach Austausch der Oberflächenchemie ein generelles Konzept zum chemischen Sensing mit aufgereinigten SWCNTs ein. 3) Die Kombination der zuvor genannten Konzepte ermöglichte die Entwicklung spezieller Nanoensoren, welche bakterielle Virulenzfaktoren wie Lipopolysaccharide (LPS), Siderophore oder sekretierte Enzyme (Protease, Nukleasen) detektieren. Mehrere dieser Sensoren wurden in einer funktionellen Hydrogelmatrix vereinigt, was ein zeitgleiches Auslesen mit Hilfe einer NIR-sensitiven Kamera ermöglichte. Mittels dieser Technik konnten typische bakterielle Infektionserreger (wie Escherichia coli, Staphylococcus aureus oder Pseudomonas aeruginosa) detektiert und ferner aufgrund ihres chemischen Fingerabdrucks unterschieden werden. 4) Zuletzt wurden Sensoren entwickelt, um Interaktionen von Pathogen mit Pflanzen zu visualisieren. Ein rationeller Ansatz ermöglichte die Detektion pflanzenstress-assoziierter Signalmoleküle wie reaktive Sauerstoffspezies (H2O2) in vivo in Arabidopsis thaliana. Darüber hinaus gelang die Detektion und Visualisierung von pflanzlichen Polyphenolen, welche als spezielle chemische Verteidigung von beispielsweise Glycine max gegen pilzliche Pathogene ausgeschüttet werden. In der vorliegenden Arbeit wurden verschiedene neue Konzepte zur spezifischen Erkennung von Biomolekülen mit SWCNTs vorgestellt und mit Hilfe aufgereinigter Nanosensoren eine hyperspektrale Detektion etabliert. Diese optimierten Sensoren ermöglichten die in vitro und in vivo Detektion von Pathogenen und deren Interaktionen, auf deren Basis in Zukunft eine verbesserte Analytik und Diagnostik im medizinischen und agrarwissenschaftlichen Bereich aufgebaut werden kann.During the global COVID-19 pandemic, the needs and benefits of fast and specific analytical tools became apparent to everyone. In particular, advances in nanotechnology promise novel healthcare diagnostics, like the identification of bacterial pathogens. However, up to now, optical nanosensors for pathogen detection rarely exist, but could pave the way for fast, label-free in situ detection of infections in the future. One class of nanomaterials with extraordinary photophysical properties are semiconducting single-walled carbon nanotubes (SWCNTs) that can serve as building blocks for such optical biosensors. These tubular carbon allotropes exhibit a diameter-dependent band gap structure, described as ‘chirality’. This leads to fluorescence emission (900-1700 nm) in the near-infrared (NIR), a spectral region most suitable for biosensing applications. To obtain functional and colloidally stable probes, the SWCNT’s hydrophobic surface needs to be non-covalently modified with biomolecules. Such SWCNT-conjugates are able to translate changes in their local chemical environment into fluorescence signals, the basic principle of optical analyte detection by SWCNT-sensors. In this thesis, consecutive steps were undertaken to tailor the functional surface chemistry and optical properties of SWCNTs for detection of pathogens and pathogen-related interactions: 1) Initially, for a better understanding of the SWCNT’s interface, a protocol to quantify adsorbed single-stranded (ss)DNA polymers was established. The calculated amount revealed several hundred DNA molecules on a single SWCNT, depending on oligonucleotide lengths and composition. This displayed the basis for further ratio-specific, bioorthogonal modifications of the nanoconjugates. 2) In addition to tailoring the surface chemistry, SWCNTs with defined emission properties for hyperspectral biosensing were isolated. One strategy made use of chirality specific SWCNT dispersions through polyfluorene polymers and further exchanged the organic interface to those needed for sensing in biological systems. The second approach generated purified samples by aqueous two-phase extraction (ATPE), and demonstrated after subsequent surface exchange a general concept for chemical sensing with chirality-pure SWCNTs. 3) Combination of these approaches facilitated the assembly of nanosensors able to detect bacterial virulence factors like lipopolysaccharides (LPS) and siderophores or secreted enzymes like proteases or nucleases. Integrated into a functional hydrogel-system and combined in an array-structure, multiple of these sensors could be read out simultaneously by a camera-assisted setup. This enabled the remote detection and discrimination of typical infection-associated bacteria (e.g. Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa), based on their chemical fingerprint. 4) Specific SWCNT sensors were developed to detect and visualize plant-pathogen interactions. In a rational approach, SWCNTs were designed to sense in vivo (Arabidopsis thaliana) reactive oxygen species (ROS, H2O2), important signaling molecules involved in plant stress response. Lastly, nanosensors for polyphenol detection were identified and used to visualize the spatiotemporal polyphenol secretion from plant roots (Glycine max), a chemical defense response after pathogen stimulus. The presented thesis introduced novel concepts to tailor the functional interface of SWCNTs for molecular recognition of important biomolecules and extends the spectral range to multiplexed approaches. These NIR-fluorescent sensors enabled detection of pathogens and pathogen interactions in vitro and in vivo, paving the way for improved healthcare- and agriculture-diagnostics.2021-10-2

Rational Design of Near-infrared Fluorescent Carbon Nanotube Biosensors with Covalent DNA-Anchors

Semiconducting single wall carbon nanotubes (SWCNTs) are versatile near infrared (NIR) fluorophores. They are non-covalently modified to create sensors that change their fluorescence when interacting with biomolecules. However, non-covalent chemistry has several limitations and prevents a consistent way to molecular recognition and reliable signal transduction. Here, we introduce a widely applicable covalent approach to create molecular sensors without impairing the fluorescence in the NIR (>1000 nm). For this purpose, we attach single-stranded DNA (ssDNA) via guanine quantum defects as anchors to the SWCNT surface. A connected sequence without guanines acts as flexible capture probe allowing hybridization with complementary nucleic acids. Hybridization modulates the SWCNT fluorescence and the magnitude increases with the length of the capture sequence (20 > 10 >> 6 bases). Incorporation of additional recognition units via this sequence enables a generic route to NIR fluorescent biosensors with improved stability. To demonstrate the potential, we design sensors for bacterial siderophores and the SARS CoV-2 spike protein. In summary, we introduce covalent guanine quantum defect chemistry as rational design concept for biosensors

Bioglass/ceria nanoparticle hybrids for the treatment of seroma: a comparative long-term study in rats.

Background: Seroma formation is a common postoperative complication. Fibrin-based glues are typically employed in an attempt to seal the cavity. Recently, the first nanoparticle (NP)-based treatment approaches have emerged. Nanoparticle dispersions can be used as tissue glues, capitalizing on a phenomenon known as 'nanobridging'. In this process, macromolecules such as proteins physically adsorb onto the NP surface, leading to macroscopic adhesion. Although significant early seroma reduction has been shown, little is known about long-term efficacy of NPs. The aim of this study was to assess the long-term effects of NPs in reducing seroma formation, and to understand their underlying mechanism. Methods: Seroma was surgically induced bilaterally in 20 Lewis rats. On postoperative day (POD) 7, seromas were aspirated on both sides. In 10 rats, one side was treated with NPs, while the contralateral side received only NP carrier solution. In the other 10 rats, one side was treated with fibrin glue, while the other was left untreated. Seroma fluid, blood and tissue samples were obtained at defined time points. Biochemical, histopathological and immunohistochemical assessments were made. Results: NP-treated sides showed no macroscopically visible seroma formation after application on POD 7, in stark contrast to the fibrin-treated sides, where 60% of the rats had seromas on POD 14, and 50% on POD 21. At the endpoint (POD 42), sides treated with nanoparticles (NPs) exhibited significant macroscopic differences compared to other groups, including the absence of a cavity, and increased fibrous adhesions. Histologically, there were more macrophage groupings and collagen type 1 (COL1) deposits in the superficial capsule on NP-treated sides. Conclusion: NPs not only significantly reduced early manifestations of seroma and demonstrated an anti-inflammatory response, but they also led to increased adhesion formation over the long term, suggesting a decreased risk of seroma recurrence. These findings highlight both the adhesive properties of NPs and their potential for clinical therapy

Sensing with Chirality Pure near Infrared Fluorescent Carbon Nanotubes

Semiconducting single wall carbon nanotubes (SWCNTs) fluoresce in the near infrared (NIR) and the emission wavelength depends on their chirality (n,m). Interactions with the environment affect the fluorescence and can be tailored by functionalizing SWCNTs with biopolymers such as DNA, which is the basis for fluorescent biosensors. So far, such biosensors were mainly assembled from mixtures of SWCNT chiralities with large spectral overlap, which affects sensitivity as well as selectivity and prevents multiplexed sensing. The main challenge to gain chirality pure sensors has been to combine approaches to isolate specific SWCNTs and generic (bio)functionalization approaches. Here, we created chirality pure SWCNT-based NIR biosensors for important analytes such as neurotransmitters and investigated the impact of SWCNT chirality/handedness as well as long-term stability and sensitivity. For this purpose, we used aqueous two-phase extraction (ATPE) to gain chirality pure (6,5)-, (7,5)-, (9,4)- and (7,6)- SWCNTs (emission at ~ 990, 1040, 1115 and 1130 nm). Exchange of the surfactant sodium deoxycholate (DOC) to specific singlestranded (ss)DNA sequences yielded monochiral sensors for small analytes (dopamine, riboflavin, ascorbic acid, pH). DOC used in the separation process was completely removed because residues impaired sensing. The assembled monochiral sensors were up to 10 times brighter than their non-purified counterparts and the ssDNA sequence affected absolute fluorescence intensity as well as colloidal (long-term) stability and selectivity for the analytes. (GT)40-(6,5)-SWCNTs displayed the maximum fluorescence response to the neurotransmitter dopamine (+140 %, Kd = 1.9 x10-7 M) and a long-term stability > 14 days. Furthermore, the specific ssDNA sequences imparted selectivity to the analytes independent of SWCNT chirality and handedness of (+/-) (6,5)-SWCNTs. These monochiral/single-color SWCNTs enabled ratiometric/multiplexed sensing of dopamine, riboflavin, H2O2 and pH. In summary, we demonstrated the assembly, characteristics and potential of monochiral (single-color) SWCNTs for multiple NIR fluorescent sensing applications

Molecular In-Depth Characterization of Chondrosarcoma for Current and Future Targeted Therapies

Chondrosarcoma (CHS) are heterogenous, but as a whole, represent the second most common primary malignant bone tumor entity. Although knowledge on tumor biology has grown exponentially during the past few decades, surgical resection remains the gold standard for the treatment of these tumors, while radiation and differentiated chemotherapy do not result in sufficient cancer control. An in-depth molecular characterization of CHS reveals significant differences compared to tumors of epithelial origin. Genetically, CHS are heterogenous, but there is no characteristic mutation defining CHS, and yet, IDH1 and IDH2 mutations are frequent. Hypovascularization, extracellular matrix composition of collagen, proteoglycans, and hyaluronan create a mechanical barrier for tumor suppressive immune cells. Comparatively low proliferation rates, MDR-1 expression and an acidic tumor microenvironment further limit therapeutic options in CHS. Future advances in CHS therapy depend on the further characterization of CHS, especially the tumor immune microenvironment, for improved and better targeted therapies

NIR-Emitting Benzene-Fused Oligo-BODIPYs for Bioimaging

Near-infrared (NIR) fluorophores are emerging tools for biophotonics because of their reduced scattering, increased tissue penetration and low phototoxicity. However, the library of NIR fluorophores is still limited. Here, we report the NIR fluorescence of two benzene-fused oligo-BODIPYs in their hexameric (H) and octameric (O) forms. These dyes emit bright NIR fluorescence (H: maxima 943/1075 nm, O: maxima 976/1115 nm) that can be excited in the NIR (H = 921 nm, O = 956 nm) or non-resonantly over a broad range in the visible region. The emission bands of H show a bathochromic shift and peak sharpening with increasing dye concentration suggesting the presence of J-aggregates. Furthermore, the emission maxima of both H and O shift up to 20 nm in solvents of different polarity. These dyes can be used as NIR ink and imaged remotely on the macroscopic level with a stand-off distance of 20 cm. We furthermore demonstrate their versatility for biophotonics by coating microscale beads and performing microrheology via NIR video particle tracking (NIR-VPT) in biopolymer (F-actin) networks. No photodamaging of the actin filaments takes place, which is typically observed for visible fluorophores and highlights the advantages of these NIR dyes

Quantum Defects in Fluorescent Carbon Nanotubes for Sensing and Mechanistic Studies

Single wall carbon nanotubes (SWCNT) fluoresce in the near infrared (NIR) and have been assembled with biopolymers such as DNA to form highly sensitive molecular sensors. They change their fluorescence when they interact with analytes. Despite the progress in engineering of these sensors the underlying mechanisms are still not understood. Here, we identify processes and rate constants that explain the photophysical signal transduction by exploiting sp3 quantum defects in the sp2 carbon lattice of SWCNTs. As a model system we use ssDNA coated (6,5)-SWCNTs, which increase their NIR emission (E11, 990 nm) up to + 250 % in response to the important neurotransmitter dopamine. In contrast, SWCNTs coated with DNA but with a low number of NO2-Aryl sp3 quantum defects decrease both their E11 (-35%) and defect related E11* emission (- 50%) at 1130 nm. Consequently, the interaction with the analyte does not change the radiative exciton decay pathway alone. Furthermore, the fluorescence response of pristine SWCNTs increases with SWCNT length, suggesting that exciton diffusion is affected. The quantum yield of pristine (6,5)-SWCNTs increases in response to the analyte from 0.6 % to 1.3 % and points to a change in non-radiative rate constants. These experimental results are explained by a Monte Carlo simulation of exciton diffusion, which supports a change of two non-radiative decay pathways together with an increase of exciton diffusion (3 rate constant model). The combination of such SWCNTs with defects and without defects enables the assembly of ratiometric sensors with opposing responses at different wavelengths. In summary, we demonstrate how perturbation of a system with quantum defects reveals the photophysical mechanism and reverses optical responses.</div