Nanoscale Tools for Biosensing and Treatment of Bacterial Biofilms and Eukaryotic Cells

Nanoparticles are well-suited for developing diagnostic and therapeutic tools due to their small size and favorable physicochemical properties. They provide unique features that many conventional approaches cannot, such as high-resolution imaging utilizing nanosensors, improved bioavailability of nanoformulated therapeutics, and targeted delivery. Polymeric nanoparticles can be easily structurally modified to tailor specific properties, making them suitable for a wide range of applications. Three studies were carried out as part of this thesis, demonstrating the diverse applications of polystyrene nanoparticles as nanosensors for biosensing in biofilms and eukaryotic cells, and as nanocarriers for the treatment of pathogenic biofilms. In the first study, a pH nanosensor based on biocompatible polystyrene nanoparticles was developed to determine and visualize the pH in biofilms. The nanosensor employs a ratiometric principle to determine pH, based on the fluorescence intensity ratio of the pH insensitive dye nile red and the pH-sensitive dye fluorescein isothiocyanate (FITC). The fluorescence is acquired by confocal laser scanning microscopy. This method allows for threedimensional measurement of pH over extended time periods, enabling detailed studies of dynamic processes in biofilms. The study demonstrated the functionality of the pH nanosensor by imaging the time-dependent pH changes induced by the metabolic activity of Escherichia coli biofilms. The nanosensor is easy to use, no special equipment is required, yet the measurements are precise, and the sensor is very robust. This is achieved by the smart design concept with its ratiometric working principle, making it a valuable tool for characterizing the chemical microenvironment of biofilms. The pH nanosensor can improve the understanding of biofilm dynamics and enable the development of improved strategies to combat biofilmassociated health problems in industry and for clinical settings. The second study describes a nanosensor for the determination of extracellular pH and the extracellular pH microenvironment of eukaryotic cells. The nanosensor operates on the same principle as the sensor in the first study, but a significant addition enables the direct measurement of pH at the cell surface. The nanosensor is conjugated to a lectin, which binds to the cell membrane and anchors the nanosensor to the cell surface. This method enables a precise and spatially resolved measurement of extracellular pH at the cell surface of individual cells. The study demonstrates the versatility and compatibility of this pH nanosensor with different cell lines from various organs, combined with effective targeting. It has great potential for studying the cellular microenvironment and gaining a deeper understanding of cellular processes based on these microenvironments. Its applications are found in biomedical research, particularly in cancer research, for understanding and studying metabolic disorders, and for diagnostic or therapeutic purposes. In the third study, a novel tool for the photodynamic eradication of biofilms was developed and applied. Polystyrene nanoparticles were used as carriers to embed the lipophilic photosensitizer (a boron-dipyrromethene derivative) and deliver it to the biofilm for activation at the target site. The study demonstrated that the photosensitizer-loaded nanoparticles were highly effective against planktonic bacteria and bacterial biofilms of pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and Streptococcus mutans. Furthermore, the study aimed to characterize the interactions between nanoparticles and biofilms to enhance the understanding of the mechanisms behind antimicrobial photodynamic therapy using nanoscale treatment agents against biofilms. The photosensitizer-loaded nanoparticles were found to be a highly effective tool in the prevention and removal of biofilms. They showed even higher efficacy than many tools in previously published studies about antimicrobial photodynamic therapy, both with and without nanoparticles. The nanoparticles presented in this study have great potential to be used as effective tools in the fight against biofilms. They offer a practical and straightforward alternative to existing methods, with a lower risk of bacterial resistance developing in the future. In summary, these studies highlight the potential of polymeric nanoparticles as carriers for effective antimicrobial treatment and as sensors for providing valuable insights into biofilm microenvironments. Furthermore, they enable precise extracellular pH measurements in diverse cell lines. These advancements hold promise for future research and applications in fields ranging from biofilm characterization to biomedical research and antimicrobial therapy

A targeted fluorescent nanosensor for ratiometric pH sensing at the cell surface

The correlation between altered extracellular pH and various pathological conditions, including cancer, inflammation and metabolic disorders, is well known. Bulk pH measurements cannot report the extracellular pH value at the cell surface. However, there is a limited number of suitable tools for measuring the extracellular pH of cells with high spatial resolution, and none of them are commonly used in laboratories around the world. In this study, a versatile ratiometric nanosensor for the measurement of extracellular pH was developed. The nanosensor consists of biocompatible polystyrene nanoparticles loaded with the pH-inert reference dye Nile red and is surface functionalized with a pH-responsive fluorescein dye. Equipped with a targeting moiety, the nanosensor can adhere to cell membranes, allowing direct measurement of extracellular pH at the cell surface. The nanosensor exhibits a sensitive ratiometric pH response within the range of 5.5–9.0, with a calculated pKa of 7.47. This range optimally covers the extracellular pH (pHe) of most healthy cells and cells in which the pHe is abnormal, such as cancer cells. In combination with the nanosensors ability to target cell membranes, its high robustness, reversibility and its biocompatibility, the pHe nanosensor proves to be well suited for in-situ measurement of extracellular pH, even over extended time periods. This pH nanosensor has the potential to advance biomedical research by improving our understanding of cellular microenvironments, where extracellular pH plays an important role

ROS generating BODIPY loaded nanoparticles for photodynamic eradication of biofilms

Bacterial biofilms can pose a serious health risk to humans and are less susceptible to antibiotics and disinfection than planktonic bacteria. Here, a novel method for biofilm eradication based on antimicrobial photodynamic therapy utilizing a nanoparticle in conjunction with a BODIPY derivative as photosensitizer was developed. Reactive oxygen species are generated upon illumination with visible light and lead to a strong, controllable and persistent eradication of both planktonic bacteria and biofilms. One of the biggest challenges in biofilm eradication is the penetration of the antimicrobial agent into the biofilm and its matrix. A biocompatible hydrophilic nanoparticle was utilized as a delivery system for the hydrophobic BODIPY dye and enabled its accumulation within the biofilm. This key feature of delivering the antimicrobial agent to the site of action where it is activated resulted in effective eradication of all tested biofilms. Here, 3 bacterial species that commonly form clinically relevant pathogenic biofilms were selected: Escherichia coli, Staphylococcus aureus and Streptococcus mutans. The development of this antimicrobial photodynamic therapy tool for biofilm eradication takes a promising step towards new methods for the much needed treatment of pathogenic biofilms

Racism, Reform, Revolution? The Segrenomics of American Education. A Book Review of \u3cem\u3eCutting School: Privatization, Segregation, and the End of Public Education\u3c/em\u3e

A review of the book Cutting School: Privatization, Segregation, and the End of Public Education, by Noliwe Rooks (The New Press, 2017)

Racism, Reform, Revolution? The Segrenomics of American Education. A Book Review of \u3cem\u3eCutting School: Privatization, Segregation, and the End of Public Education\u3c/em\u3e

A review of the book Cutting School: Privatization, Segregation, and the End of Public Education, by Noliwe Rooks (The New Press, 2017)

Detection of metastable electronic states by Penning trap mass spectrometry

State-of-the-art optical clocks achieve fractional precisions of $10^{-18}$ and below using ensembles of atoms in optical lattices or individual ions in radio-frequency traps. Promising candidates for novel clocks are highly charged ions (HCIs) and nuclear transitions, which are largely insensitive to external perturbations and reach wavelengths beyond the optical range, now becoming accessible to frequency combs. However, insufficiently accurate atomic structure calculations still hinder the identification of suitable transitions in HCIs. Here, we report on the discovery of a long-lived metastable electronic state in a HCI by measuring the mass difference of the ground and the excited state in Re, the first non-destructive, direct determination of an electronic excitation energy. This result agrees with our advanced calculations, and we confirmed them with an Os ion with the same electronic configuration. We used the high-precision Penning-trap mass spectrometer PENTATRAP, unique in its synchronous use of five individual traps for simultaneous mass measurements. The cyclotron frequency ratio $R$ of the ion in the ground state to the metastable state could be determined to a precision of $\delta R=1\cdot 10^{-11}$, unprecedented in the heavy atom regime. With a lifetime of about 130 days, the potential soft x-ray frequency reference at $\nu=4.86\cdot 10^{16}\,\text{Hz}$ has a linewidth of only $\Delta \nu\approx 5\cdot 10^{-8}\,\text{Hz}$, and one of the highest electronic quality factor ($Q=\frac{\nu}{\Delta \nu}\approx 10^{24}$) ever seen in an experiment. Our low uncertainty enables searching for more HCI soft x-ray clock transitions, needed for promising precision studies of fundamental physics in a thus far unexplored frontier

Development of the IntCal database

The IntCal family of radiocarbon (14C) calibration curves is based on research spanning more than three decades. The IntCal group have collated the 14C and calendar age data (mostly derived from primary publications with other types of data and meta-data) and, since 2010, made them available for other sorts of analysis through an open-access database. This has ensured transparency in terms of the data used in the construction of the ratified calibration curves. As the IntCal database expands, work is underway to facilitate best practice for new data submissions, make more of the associated metadata available in a structured form, and help those wishing to process the data with programming languages such as R, Python, and MATLAB. The data and metadata are complex because of the range of different types of archives. A restructured interface, based on the “IntChron” open-access data model, includes tools which allow the data to be plotted and compared without the need for export. The intention is to include complementary information which can be used alongside the main 14C series to provide new insights into the global carbon cycle, as well as facilitating access to the data for other research applications. Overall, this work aims to streamline the generation of new calibration curves

Environmental considerations and current status of grouping and regulation of engineered nanomaterials

This article reviews the current status of nanotechnology with emphasis on application and related environmental considerations as well as legislation. Application and analysis of nanomaterials in infrastructure (construction, building coatings, and water treatment) is discussed, and in particular nanomaterial release during the lifecycle of these applications. Moreover, possible grouping approaches with regard to ecotoxicological and toxicological properties, and the fate of nanomaterials in the environment are evaluated. In terms of potential exposure, the opportunities that arise from leveraging advances in several key areas, such as water treatment and construction are addressed. Additionally, this review describes challenges with regard to the European Commission’s definition of ‘nanomaterial’. The revised REACH information requirements, intended to enable a comprehensive risk assessment of nanomaterials, are outlined

Analytical and toxicological aspects of nanomaterials in different product groups: Challenges and opportunities

The widespread integration of engineered nanomaterials into consumer and industrial products creates new challenges and requires innovative approaches in terms of design, testing, reliability, and safety of nanotechnology. The aim of this review article is to give an overview of different product groups in which nanomaterials are present and outline their safety aspects for consumers. Here, release of nanomaterials and related analytical challenges and solutions as well as toxicological considerations, such as dose-metrics, are discussed. Additionally, the utilization of engineered nanomaterials as pharmaceuticals or nutraceuticals to deliver and release cargo molecules is covered. Furthermore, critical pathways for human exposure to nanomaterials, namely inhalation and ingestion, are discussed in the context of risk assessment. Analysis of NMs in food, innovative medicine or food contact materials is discussed. Specific focus is on the presence and release of nanomaterials, including whether nanomaterials can migrate from polymer nanocomposites used in food contact materials. With regard to the toxicology and toxicokinetics of nanomaterials, aspects of dose metrics of inhalation toxicity as well as ingestion toxicology and comparison between in vitro and in vivo conclusions are considered. The definition of dose descriptors to be applied in toxicological testing is emphasized. In relation to potential exposure from different products, opportunities arising from the use of advanced analytical techniques in more unique scenarios such as release of nanomaterials from medical devices such as orthopedic implants are addressed. Alongside higher product performance and complexity, further challenges regarding material characterization and safety, as well as acceptance by the general public are expected

Monitoring and imaging pH in biofilms utilizing a fluorescent polymeric nanosensor

Biofilms are ubiquitous in nature and in the man-made environment. Given their harmful effects on human health, an in-depth understanding of biofilms and the monitoring of their formation and growth are important. Particularly relevant for many metabolic processes and survival strategies of biofilms is their extracellular pH. However, most conventional techniques are not suited for minimally invasive pH measurements of living biofilms. Here, a fluorescent nanosensor is presented for ratiometric measurements of pH in biofilms in the range of pH 4.5–9.5 using confocal laser scanning microscopy. The nanosensor consists of biocompatible polystyrene nanoparticles loaded with pH-inert dye Nile Red and is surface functionalized with a pH-responsive fluorescein dye. Its performance was validated by fluorometrically monitoring the time-dependent changes in pH in E. coli biofilms after glucose inoculation at 37 °C and 4 °C. This revealed a temperature-dependent decrease in pH over a 4-h period caused by the acidifying glucose metabolism of E. coli. These studies demonstrate the applicability of this nanosensor to characterize the chemical microenvironment in biofilms with fluorescence methods