Nanosensor Detection of an Immunoregulatory Tryptophan Influx/Kynurenine Efflux Cycle

Mammalian cells rely on cellular uptake of the essential amino acid tryptophan. Tryptophan sequestration by up-regulation of the key enzyme for tryptophan degradation, indoleamine 2,3-dioxygenase (IDO), e.g., in cancer and inflammation, is thought to suppress the immune response via T cell starvation. Additionally, the excreted tryptophan catabolites (kynurenines) induce apoptosis of lymphocytes. Whereas tryptophan transport systems have been identified, the molecular nature of kynurenine export remains unknown. To measure cytosolic tryptophan steady-state levels and flux in real time, we developed genetically encoded fluorescence resonance energy transfer nanosensors (FLIPW). The transport properties detected by FLIPW in KB cells, a human oral cancer cell line, and COS-7 cells implicate LAT1, a transporter that is present in proliferative tissues like cancer, in tryptophan uptake. Importantly, we found that this transport system mediates tryptophan/kynurenine exchange. The tryptophan influx/kynurenine efflux cycle couples tryptophan starvation to elevation of kynurenine serum levels, providing a two-pronged induction of apoptosis in neighboring cells. The strict coupling protects cells that overproduce IDO from kynurenine accumulation. Consequently, this mechanism may contribute to immunosuppression involved in autoimmunity and tumor immune escape

Smartphone-based analytical devices with optical detection for on-site biosensing: environmental, food and forensic applications

There has been an increasing demand for fast and easy monitoring technologies designed to respond to different analytes. The standard analytical techniques offer accurate and precise results; however, they require clean samples, sophisticated equipment and skilled personnel. For these reasons, they are not suitable for on site, real-time, cost-effective routine monitoring. Biosensors are analytical devices integrating a biological recognition element and a transducer element able to convert the biological response into an easily measurable analytical signal. These tools can easily quantify an analyte or a class of analytes of interest even in a complex matrix, like clinical or environmental samples, thanks to the specificity of the biological components and can be easily implemented in portable devices. The activity carried out during my PhD was mainly focused on the development of different portable paper-based biosensors for multianalyte detection and their implementation into portable analytical devices for point-of-care and point-of-need applications. In particular, enzymes and cells (bacteria and mammalian cell) have been exploited as biorecognition elements, in some cases even by coupling different elements in the same biosensor to increase its robustness. The final goal of biosensors developed was the application in the environmental and forensic fields, since the target analytes are organophosphorus pesticides, heavy metals and molecules with androgenic activity, including new drugs or endocrine disrupting chemicals. Moreover, different optical detection principles (chemiluminescence, bioluminescence, colorimetry) have been exploited and coupled to create an orthogonal detection, which provides more accurate results. Different portable detectors, such as coupled-charged device, smartphone cameras and silicon photomultiplier, and also benchtop laboratory instruments have been used to validate and support the developed biosensors. Several paper-based platforms have been designed and implemented with adaptors and devices fabricated using a dual-extrusion 3D printer to better adapt to the type of assay, reagents, samples and detection method

Trehalose Transport in Corynebacterium glutamicum and its Significance for Cell Envelope Synthesis

Corynebacterium glutamicum ist ein Gram-positives, apathogenes Bodenbakterium. Es ist ein wichtiger industriell genutzter Aminosäureproduzent und dient als Modellorganismus für die Synthese der Zellhülle in verwandten pathogenen Arten wie Mycobacterium tuberculosis, dem Tuberkuloseerreger. Ein auffälliges Merkmal der Zellhülle dieser Bakterien ist das Vorhandensein einer zweiten Per¬meabilitätsbarriere ähnlich der äußeren Membran Gram-negativer Bakterien. Wichtige Bestandteile sind Trehalosemono- (TMM) und dimycolat (TDM), langkettige Fettsäuren, die mit einem Molekül des Disaccharids Trehalose verestert sind. Frühere Versuche mit C. glutamicum zeigten, dass die Verknüpfung im Periplasma erfolgt, wofür der Export beider Moleküle notwendig ist. Dieses Modell wurde durch neue Studien in Frage gestellt, welche MmpL-Transporter mit dem Export von TMM aus dem Cytosol in Verbindung brachten. Um das beschriebene Modell der TMM-Synthese zu überprüfen, wurde daher in dieser Arbeit der Export von Trehalose untersucht. In einem C. glutamicum Teststamm konnten die cytosolische Umwandlung zuvor aufgenommener Maltose in Trehalose und dessen Exkretion nachgewiesen werden, wobei eine extrazelluläre Um¬wandlung ausgeschlossen wurde. Die bestimmte Exportrate von 0.19 nmol × mg-1 cdw × min-1 ist für die TMM-Synthese während des Wachstums ausreichend und war unabhängig von der Aktivität mechanosensitiver Kanäle, welche Trehalose in anderen Organismen freisetzen, und von der TMM-Synthese. Zusammen mit mechanis¬tischen Analysen der Exkretion deutet dies auf das Vorhandensein eines Trehalosetransporters hin, der diese für die TMM-Synthese im Periplasma bereitstellt. TMM und TDM wurde nur in Zellen nachgewiesen, in denen einer von zwei redundanten MmpL-Transportern aktiv war. Es wird daher ein Modell vorgeschlagen, das eine periplasmatische Synthese von TMM aus zuvor exportierten Substraten und einem MmpL-vermittelten Transport von TMM aus der äußeren Schicht der Plasmamembran zur Mycolatschicht vereint. Da ein rationaler Ansatz nicht zur Identifizierung eines Trehaloseexporters führte, wurde ein genetisch kodierter Trehalosesensor entwickelt und optimiert, welcher für das Screening einer Mutantenbibliothek eingesetzt werden sollte. Die Affinität dieses Sensors konnte jedoch durch gerichtete Mutagenese nicht ausreichend reduziert werden, um eine in vivo-Applikation zu ermöglichen. Der Trehaloseexporter von C. glutamicum ist daher weiterhin unbekannt

Molecular Probes, Chemosensors, and Nanosensors for Optical Detection of Biorelevant Molecules and Ions in Aqueous Media and Biofluids

Synthetic molecular probes, chemosensors, and nanosensors used in combination with innovative assay protocols hold great potential for the development of robust, low-cost, and fast-responding sensors that are applicable in biofluids (urine, blood, and saliva). Particularly, the development of sensors for metabolites, neurotransmitters, drugs, and inorganic ions is highly desirable due to a lack of suitable biosensors. In addition, the monitoring and analysis of metabolic and signaling networks in cells and organisms by optical probes and chemosensors is becoming increasingly important in molecular biology and medicine. Thus, new perspectives for personalized diagnostics, theranostics, and biochemical/medical research will be unlocked when standing limitations of artificial binders and receptors are overcome. In this review, we survey synthetic sensing systems that have promising (future) application potential for the detection of small molecules, cations, and anions in aqueous media and biofluids. Special attention was given to sensing systems that provide a readily measurable optical signal through dynamic covalent chemistry, supramolecular host–guest interactions, or nanoparticles featuring plasmonic effects. This review shall also enable the reader to evaluate the current performance of molecular probes, chemosensors, and nanosensors in terms of sensitivity and selectivity with respect to practical requirement, and thereby inspiring new ideas for the development of further advanced systems

Self-assembling, coiled coil interfaces for nanoscale amperometric biosensors

Both gold nanostructures and carbon nanotubes are rapidly emerging platforms for amperometric biosensor detection. Homogeneous display of biomolecular ligands is a key process in forming nanoscale array biosensors, improving the chances for sufficient signal strength and reproducibility at the limits of sensor geometries. The use of selfassembling interfaces on gold electrodes and CNT arrays enables homogeneous display, solvent access, target capture, sensor surface regeneration and potential for molecular wiring. Our approach uses an anchor peptide, covalently linked to the sensor surface, that is able to non-covalently capture probe labeled targets in solution with a high affinity and minimal surface leaching. The use of non-disrupting, metal-chelating scaffolds within the coiled coil sequence was tested for molecular wiring capabilities using model redox targets. Recombinant cassette production of probe sequences was investigated as a means to enable control over spatial and stoichiometric placement of redox active mediators and/or biomolecules. This thesis defines potential challenges that face protein interfacing in amperometric biosensors as well as opportunities for future developments.Ph.D., Biomedical Engineering -- Drexel University, 200

An inert continuous microreactor for the isolation and analysis of a single microbial cell

Studying biological phenomena of individual cells is enabled by matching the scales of microbes and cultivation devices. We present a versatile, chemically inert microfluidic lab-on-a-chip (LOC) device for biological and chemical analyses of isolated microorganisms. It is based on the Envirostat concept and guarantees constant environmental conditions. A new manufacturing process for direct fusion bonding chips with functional microelectrodes for selective and gentle cell manipulation via negative dielectrophoresis (nDEP) was generated. The resulting LOC system offered a defined surface chemistry and exceptional operational stability, maintaining its structural integrity even after harsh chemical treatment. The microelectrode structures remained fully functional after thermal bonding and were proven to be efficient for single-cell trapping via nDEP. The microfluidic network consisted solely of glass, which led to enhanced chip reusability and minimized interaction of the material with chemical and biological compounds. We validated the LOC for single-cell studies with the amino acid secreting bacterium Corynebacterium glutamicum. Intracellular l-lysine production dynamics of individual bacteria were monitored based on a genetically encoded fluorescent nanosensor. The results demonstrate the applicability of the presented LOC for pioneering chemical and biological studies, where robustness and chemically inert surfaces are crucial parameters for approaching fundamental biological questions at a single-cell level

ENGINEERED ACTIVITY SENSORS FOR PREDICTIVE IMMUNE MONITORING

Immunotherapies are transforming the treatment of immunological disorders for patients with intractable diseases, for instance through the activation of anti-tumor immunity or the suppression of host reactivity against organ transplants. However, modest response rates and treatment resistance remain clinical barriers, driving efforts to improve response monitoring to better guide clinical decision-making. Most current standards to assess immunotherapy responses rely on evaluation of disease burden by either the core biopsy (e.g., to detect transplant rejection) or radiographic imaging (e.g., to assess tumor regression), yet these approaches primarily focus on morphological features downstream of the immune response. There remains a need for early on-treatment biomarkers to identify patients that may benefit from treatment continuation, alleviate the risks of immune-mediated toxicity, and provide opportunities to treat resistant patients with alternative therapies. Biomarkers of T cell immunity have the potential to monitor the onset of therapeutic responses as elevation of T cell activity in the tumor microenvironment drives tumor control, and suppression of host T cell reactivity towards donor cells promotes transplant tolerance. Proteases are important mediators of immunity and diseases, providing an opportunity to predict responses to immunotherapy early on-treatment. Of note, T cell killing occurs via the classic perforin and granzyme-mediated pathway – the latter of which comprises a family of potent serine proteases – while proteases like matrix-degrading and inflammatory proteases are implicated in major disease hallmarks such as angiogenesis and inflammation. In this thesis, I engineer activity sensors of T cell immunity for two important clinical problems: detecting transplant rejection and monitoring tumor responses during immunotherapy. These sensors monitor the activity of proteases during T cell responses and produce a remote readout in urine. I first develop activity-based nanosensors monitoring granzyme B (GzmB) as noninvasive biomarkers of T cell-mediated acute transplant rejection. Using a skin graft mouse model of organ transplantation, I demonstrate that GzmB nanosensors detect the onset of rejection and indicate allograft failure in recipients treated with subtherapeutic immunosuppression. Then, to noninvasively assess response and resistance to cancer immunotherapy, I design ImmuNe Sensors for monItorinG cHeckpoint blockade Therapy (INSIGHT) by conjugating activity sensors to checkpoint antibodies (e.g., αPD1). In tumor models of immune checkpoint blockade (ICB) response, I show that αPD1-GzmB sensor conjugates retain therapeutic efficacy while producing increased urine signals indicative of early on-treatment responses. Additionally, a multiplexed INSIGHT library sensing tumor and immune proteases enables the development of machine learning classifiers based on urinary outputs to accurately stratify two mechanisms of ICB resistance. This thesis motivates the development of in vivo immune monitoring technologies to maximize the precision and benefit of immunotherapy.Ph.D

Novel in vivo biosensors for monitoring of mammalian cell cultures

Mammalian cell cultures are used for production of biopharmaceuticals, e.g. monoclonal antibodies. Only mammalian hybridoma cells contain the pathways for antibody production, but due to their multicellular origin the cells have complex nutrient requirements. Cell growth and antibody production are limited by supply of essential nutrients such as glutamine and accumulation of toxic waste products such as lactate. Many attempts have been made at tackling these challenges, e.g. by optimising growth media to keep metabolite concentrations at optimal levels. These approaches have been hampered by our ability to monitor relevant cell culture parameters such as metabolite concentration dynamics in real time. The aim of this study is to develop a solution to this problem using a synthetic biology approach. Whole-cell bacterial biosensors for important culture parameters, glutamine, leucine, alanine and lactate, were designed, built and characterised. The biosensors were designed from natural metabolite-sensing systems, specifically the Escherichia coli Ntr regulon, Lrp regulon and lldPRD operon and the Bacillus subtilis GlnK-GlnL system. Characterisation of the biosensors in defined medium using known lactate concentrations was followed by validation in mammalian cell culture media and using cell culture samples. A lactate sensor based on the lldPRD operon showed a reliable lactate-response during initial characterisation and was chosen to determine lactate concentrations in cell culture samples in parallel with lactate analysis using a bioprofiler. Generally, the lactate concentrations from the two methods showed a good match. Data points where the results differed showed that there are some sources of error in the usage of the biosensor that could be addressed in future. The results of this study also highlight the many challenges of applying synthetic biology constructs to complex industrial contexts. The biosensors presented in this study are more generally applicable in any experimental context that requires sensing of metabolites.Open Acces