Utilization of Nanoparticles for Photoacoustic Chemical Imaging

Tumors are known to have unique chemical properties, such as low pH (acidosis), high K+ (hyperkalemia), and low O2 (hypoxia). Tumor acidosis has been known to influence therapeutic activities of chemotherapeutic drugs. Another conventional cancer treatment, radiation therapy, is highly dependent on local oxygen concentrations. Hyperkalemia has been recently reported to suppress the immune response of activated T-cells. It is also believed that the spatial distribution of these analytes and its heterogeneity, are of relevance. Despite the importance of such chemical information on tumors, there are no clinically available tools for “quantitative” pH, K+, or tissue O2 imaging. Here, photoacoustic (PA) imaging is employed to provide chemical imaging of all these target analytes for cancer (pH, O2 and K+). As for pH, we report on an in vivo pH mapping nanotechnology. This subsurface chemical imaging is based on tumor-targeted, pH sensing nanoprobes and multi-wavelength photoacoustic imaging (PAI). The nanotechnology consists of an optical pH indicator, SNARF-5F, 5-(and-6)-Carboxylic Acid, encapsulated into polyacrylamide nanoparticles with surface modification for tumor targeting. Facilitated by multi-wavelength PAI plus a spectral unmixing technique, the accuracy of pH measurement inside the biological environment is not susceptible to the background optical absorption of biomolecules, i.e., hemoglobins. As a result, both the pH levels and the hemodynamic properties across the entire tumor can be quantitatively evaluated with high sensitivity and high spatial resolution in in vivo cancer models. For K+, we extend this technique to ion-selective photoacoustic optodes (ISPAOs) that serve at the same time as fluorescence-based ISOs, and apply it specifically to potassium (K+). However, unfortunately, sensors capable of providing potassium images in vivo are still a future proposition. Here, we prepared an ion-selective potassium nanosensor (NS) aimed at in vivo photoacoustic (PA) chemical imaging of the extracellular environment, while being also capable of fluorescence based intracellular ion-selective imaging. This potassium nanosensor (K+ NS) modulates its optical properties (absorbance and fluorescence) according to the potassium concentration. The K+ NS is capable of measuring potassium, in the range of 1 mM to 100 mM, with high sensitivity and selectivity, by ISPAO based measurements. Also, a near infrared dye surface modified K+ NS allows fluorescence-based potassium sensing in the range of 20 mM to 1 M. The K+ NS serves thus as both PA and fluorescence based nanosensor, with response across the biologically relevant K+ concentrations, from the extracellular 5 mM typical values (through PA imaging) to the intracellular 150 mM typical values (through fluorescence imaging). Lastly, nano-enabled tissue O2 monitoring by PA, called lifetime-based PA (PALT) imaging, was introduced and demonstrated. A known PALT oxygen indicator, Oxyphor G2, is conjugated into polyacrylamide nanoparticles, called G2-PAA NP. The oxygen sensing capability of the G2-PAA NP has been confirmed in vitro and in vivo studies. In an Appendix, we show how to monitor photodynamic therapy (PDT) using the PALT approach to measure the local oxygen depletion as a function of PDT time. Oxygen depletion during PDT is monitored using both oximeter and PALT spectroscopy in vitro. The latter is enabled by theranostic NPs of methylene blue (MB) conjugated PAA, used for both PALT and PDT. This synergistic approach has good potential for personalized medicine.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/143924/1/lechang_1.pd

Development of nanocatalytic-based assay for the detection of an endocrine disrupting compound in aqueous solution

Endocrine disrupting compound (EDC) pollutants raise a concern among researchers as these pollutants are implicated in the increasing incidence of testicular, breast and thyroid cancers. Some of these chemicals are widely used for plastics production and discharged into the water system as industrial effluents that could harm the ecosystem as well as plant, animal and human life. Thus, rapid detection and quantification of EDCs in water is desired for screening and investigative purposes. For this purpose, nanoparticle-based methods appear to be potentially efficient, quick and cost-effective techniques to rapidly assess this toxic pollutant. The main focus of this study was to synthesize heterogeneous nanoparticles, iron oxide/gold nanoparticles (IONPs/AuNPs) and to manipulate their synergistic effects for the development of a nanoparticles-based assay, specifically for the EDC compound, 17β-estradiol. As the first step, IONPs and AuNPs were synthesized separately and heterogeneous nanoparticles were formed by a simple electrostatic- self- assembly technique. The unique physiochemical properties of this hybrid nanoparticle were investigated as a supporting material for biomolecules, as well for its intrinsic peroxidase-like activity using a hydrogen peroxidase dependent system. The formation of the IONPs/AuNPs was verified using several characterization tools such as UV-Vis spectrophotometry, Dynamic Light Scattering (DLS), Transmission Electron Microscope (TEM), Energy Dispersive X-ray (EDX) and X-ray Photoelectron Spectroscopy (XPS). The diameter calculated from TEM was 16.1 ± 11.1 nm and EDX confirmed the presence of the Fe and Au elements. From a heterostructural analysis using HRTEM and XPS data, an alloy-like morphology (Fe/Au) was suggested for the heterogeneous nanoparticles, rather than a core-shell structure. The Fe/Au nanoparticles showed good potential for the basis of a colorimetric assay for glucose detection using glucose oxidase immobilized on the Fe/Au surface. In addition, the Fe/Au nanoparticles also showed a significant peroxidase-like activity. A nanocatalytic-based assay was developed by modifying the nanoparticles surface with an aptamer in order to specifically “capture” the target molecule, 17β-estradiol. The formation of a Fe/Au-17β-estradiol complex significantly hampered the peroxidase-like catalytic activity resulting in the development of a unique nanosensor system based on the extent of loss of peroxidase activity. Development of the nanocatalytic-based assay suggests the potential application of Fe/Au nanoparticles to capture, separate and detect a selective target as well as a basis for the development of a rapid, simple and reliable detection tool. The heterogeneous Fe/Au nanoparticles show a remarkable synergistic property for application in nanosensor system. Therefore, some of the work presented here can be extended in certain major directions such as heterostructure formation and optimization of nanocatalytic-based assay

Single Molecule Particle Analysis using Nanotechnology

Nanotechnology is the area of science that involves creation of devices/materials or systems in the nanometer scale. The last few decades have seen an increasing demand for rapid, sensitive, and cheaper diagnostic tools in healthcare. Advances in fabrication technologies have led to more miniaturized systems that are satisfying the promise of “micro total analysis” or “lab-on-chip” systems by facilitating the integration of multiple processing steps into a single device or multiple task-specific devices into a fluidic motherboard (i.e., modular microfluidics). The field of nanotechnology has the ability to revolutionize medical diagnostics by facilitating point-of-care testing with greater sensitivity even at the single molecule level. This allows for the screening of diseases at an early stage by identifying biomarkers of the diseases that are in extremely low concentrations in the blood (i.e., liquid biopsy). To this realization, we have used thermoplastics as our choice of material to fabricate microfluidic/nanofluidic hybrid systems that can evaluate how well a patient responds to chemotherapy, identify single nucleotide polymorphisms that cause major life threatening diseases such as stroke and caner, and development of nanofluidic devices to enumerate SARS CoV-2 viral particles that causes the novel coronavirus of 2019. We developed a high-throughput nanofluidic circuit on which single DNA molecules can be stretched to near their full contour length in nanochannels (<100 nm). Patients with cancer undergoing chemotherapy have more oxidative damage in their DNA compared to a healthy individual, which is an indicator of their response to therapy. We tested the device using calf thymus DNA standards labelled with a bis-intercalating dye and the abasic sites were labelled with another dye. Thus, the DNA molecules that were stretched in the nanochannels were parked and visualized using a fluorescent microscope. The abasic sites that were labelled were identified with their position in the DNA and the number of abasic sites per 105 nucleotides identified. This technique can be effectively used on samples having mass limits (picograms range) and where PCR cannot be utilized. Higher the number of abasic sites, better the response of the patient to chemotherapy, such as doxorubicin for breast cancer patients. While this nanofluidic circuit was used only to visualize the abnormalities in DNA, the next device we developed, called the nanosensor, facilitates the integration of multiple processes into a single device. The nanosensor was used to identify point mutations in DNA or mRNA responsible for diseases such as cancer and stroke, respectively. The device featured 8 pixel array populated with 1 µm pillars, which act as a solid support for Ligase Detection Reactions (spLDR) that can identify a single nucleotide mutation in a DNA from a large majority of wild type DNA. The spLDR can also identify mRNA transcripts from the design of spLDR primers that specifically recognize a unique transcript. The reaction is performed on the pixel arrays and the products are subsequently shuttled into nanometer flight tubes featuring two in-plane nanopores that act as resistive pulse sensors (RPS) to generate a current drop as the products pass through these pores. The time-of-flight (TOF) between the pores in series are used to distinguish between normal and mutated DNA, thus acting as a diagnostic appropriate for the precision medicine initiative. We were able to successfully fabricate the device, run COMSOL simulations to test operation using both hydrodynamic and electrokinetic flows, which were verified via experimentation to establish the functionality of the device to perform the above mentioned processes. The hydrodynamic flow operations used for spLDR was tested using Rhodamine B and the electrokinetic flow to inject the products of the spLDR into the flight tube was tested using oligonucleotides (25mer). Further, plastic-based nanofluidic devices were extended to detect the presence of SARS-CoV-2 viral particles using a nanopore of 350 nm in effective diameter, which has called a nano-coulter Counter (nCC). Briefly, saliva samples containing the viral particles were run through a microfluidic affinity chip containing pillars with surface-immobilized aptamers specific to the SARS-CoV-2 particles. The captured viral particles were released from the microfluidic chip using a blue light and the elute containing only the SARS viral particles were sent to the nCC, which used the RPS technique to count the number of particles. We designed multiple iterations of the nCC and used COMSOL simulations to guide device development. Using the combined principle of hydrodynamic and electrokinetic flow to introduce the viral particles into the nCC, we were able to detect patients with COVID-19 as well as estimate the viral load in SARS CoV-2 standards based on the frequency of the signals generated by correlating the results to a calibration curve. Thus, this combined multi-chip process can diagnose COVID-19 in <20 min thus venturing as an in-home diagnostic kit in the future by automating the operations into a hand-held device

Surface-enhanced Raman Spectroscopy for Single Molecule Analysis and Biological Application

Surface-enhanced Raman spectroscopy (SERS) is a surface analytical technique, which enhances the Raman signal based on the localized surface plasmon resonance (LSPR) phenomenon. It has been successfully used for single molecule (SM) detection and has extended SERS to numerous applications in biomolecular detection. However, SM detection by SERS is still challenging especially with traditional SERS substrates and detection methods. In addition, the fundamental understanding of the SERS enhancement mechanism is still elusive. Furthermore, the application of SERS in biological field is still in the early stage. To address these challenges, there are two main aspects of SERS studied in my dissertation: (a) fundamental aspects through systematic experimental studies combined with simulations, which focus on SM detection, Raman enhancement mechanisms, and (b) the development and optimization of the SERS-based nanoprobe for biomarkers detection from fluidic devices to a single cell. In my dissertation, the following studies have been investigated. First, the sensitivity of a home-made SERS instrument was tested. SM detection was realized by utilizing a highly curved nanoelectrode (NE) to limit the number of attached nanoparticle (NP), which will allow us to have even a single NP on NE (NPoNE) junction in the SERS detection area. The molecule number in a single NPoNE junction which contributes to SERS can be hundreds or even SM. In this first study, we also conducted a correlation study between electrochemical current and SERS to monitor the dynamic formation of the plasmonic junctions. Second, we investigate electromagnetic and chemical enhancement factor tuning by the electrode potential with the assistant of Au@Ag core-shell NPs. The electrode potential induced electromagnetic enhancement (EME) tuning in the Au@Ag NPoNE structure has been confirmed by 3D Finite-difference time-domain (FDTD) simulations. Last is the design of a SERS-based nanoprobe for biomarkers detection and the effort towards single cell analysis. Finally, several SERS-active substrates were examined for biomarkers (H+, glucose, and H2O2) detection, including gold NPs (AuNPs) colloid and AuNPs decorated glass nanopipette. In summary, my dissertation presents the fabrication and development of gold tip nanoelectrode for chemical detection, which can achieve SM sensitivity. SM SERS can be used to improve the fundamental understanding and provide more in-depth insight into mechanisms of SERS and the chemical behaviors of SM on surfaces and in plasmonic cavities. Second, the fabrication and optimization of SERS-active, flexible nanopipette for biological applications. The flexible nanopipette probe provides a platform for reliable detection and quantitative analysis of biomarkers at a single cell level, which is critical and vital for detecting diseases earlier and understanding the fundamental biological process better

Targeted fluorescent optical nanosensors for imaging and measuring function in intracellular microdomains

Nanosensors offer the opportunity to measure intracellular domains with minimal chemical or physical perturbation. Typically only 60 nm in diameter and synthesised from polymer matrices they entrap chemical sensing elements and can be surface functionalised allowing for further chemical modification. Upon intracellular localisation the cellular environment can be monitored using conventional techniques such as confocal laser scanning microscopy. Reported here is one use of nanosensors to investigate the mechanisms of intracellular delivery mediated via the cell penetrating peptide, Tat. It is shown that information obtained from the nanosensors reveals that the post-delivery environment is representative of a lysosome in terms of both pH and morphology. The delivery mechanism of Tat, however, is shown to be dependent upon the cargo being delivered, corresponding to the absence or presence of a body of polymer matrix; thus nanosensors have been used to further the understanding of the cell penetrating mechanisms of Tat peptide. Technological aspects of nanosensor development have been investigated including polymer matrix modification and different methods of incorporating fluorophores into the nanosensor body. Addressing nanosensors located in an intracellular domain has historically been achieved with epi-fluorescent and confocal microscopy acquiring data from individual or low numbers of cells only. Reported here is the combination of nanosensors with flow cytometry as a technique for en masse investigation into entire cell populations

Optophysiologie SERS : analyse in vitro d’environnement cellulaire en Raman exalté par les surfaces

Afin d’assurer la communication et la régulation de leur métabolisme, les cellules vivantes sécrètent une panoplie de petites et grandes molécules agissant comme messagers chimiques. Ces messagers sont généralement libérés dans l’environnement extracellulaire et diffusent jusqu’à l’atteinte d’une cible moléculaire bien précise. Cet évènement de reconnaissance biologique induit une cascade de réactions biochimiques qui sont le fondement des fonctions biologiques des cellules. Un dysfonctionnement associé à la sécrétion de messagers chimiques est associé à plusieurs conséquences cliniques. Par exemple, un changement de composition des neurotransmetteurs lors de la libération synaptique dans le cerveau est associé à la maladie d’Alzheimer, la maladie de Parkinson, la dépression, les dépendances et la schizophrénie. L’analyse des messagers chimiques est donc d’une grande importance dans le domaine clinique. Néanmoins, les méthodes analytiques contemporaines ne sont pas aptes à mesurer les changements rapides et hétérogènes des messagers chimiques lors d’évènements de sécrétion cellulaire. De nouvelles techniques permettant une analyse hautement sensible de plusieurs messagers chimiques simultanément et de manière localisée sont nécessaires. Les matériaux plasmoniques permettent l’utilisation de spectroscopies exaltée par les métaux comme la diffusion Raman exaltée par les effets de surfaces (SERS). Grâce à la spécificité moléculaire du SERS et à sa haute sensibilité, pouvant atteindre la détection d’une seule molécule, les capteurs SERS offrent divers avantages permettant le développement de nouvelles technologies dédiées aux mesures des sécrétions cellulaires. L’objectif principal de la thèse est la mise au point de nouveaux capteurs SERS permettant l’analyse des sécrétions cellulaires avec une haute sensibilité et une haute sélectivité. Pour y arriver, des nanoparticules d’or furent adsorbées sur des capillaires de verres nanométriques de type patch clamp. L’immobilisation des nanoparticules sur ces patch clamp génère un nanocapteur plasmonique pouvant être positionné dans l’espace, tout en conservant un état d’agrégation constant, facteur clé en SERS. Ces nanocapteurs furent donc positionnés près de cellules in vitro, puis des spectres SERS furent acquis dans le temps. Afin d’analyser cette réponse optique complexe, de nouvelles méthodes chimiométriques ont été développées. Comme première approche, un code-barres unique fut extrait à partir de chaque parton vibrationnel expérimental mesuré par SERS dans le temps, puis comparés à ceux d’une librairie extraient à partir de standards. Cette comparaison permet : (1) l’identification de la molécule selon son code-barres et (2) une analyse semi-quantitative en dénombrant le nombre de fois qu’un code-barres fut identifié sur une période de temps précise. Cette méthode chimiométrique, définie comme l’optophysiologie SERS, fut employée pour sonder les métabolites extracellulaires sécrétés par des cellules MDCKII au niveau basal et post-stimulation. Par sa nouveauté, l’optophysiologie requiert une optimisation des paramètres d’analyse, limitant sa capacité de multiplexage et son adaptation pour la détection de nouvelles cibles moléculaires. Afin d’en améliorer la sensibilité et la sélectivité, un nouvel algorithme d’évaluation des codes-barres fut implémenté à l’optophysiologie. Ce nouvel algorithme fut employé pour sonder les neurotransmetteurs sécrétés par des neurones dopaminergiques originaires du mésencéphale de souris. De plus, afin de visualiser les neurones et positionner correctement les nanocapteurs près des varicosités axonales, sites de libération majeure des neurotransmetteurs, un microscope combiné permettant l’imagerie de fluorescence et la spectroscopie Raman fut construit. À l’aide de ces outils, il fut possible de mesurer les changements relatifs de concentration in vitro de cinq neurotransmetteurs dans des conditions basales et durant plusieurs cycles de stimulation. Les performances analytiques de l’optophysiologie SERS dépendent, majoritairement, des codes-barres utilisés pour l’analyse, nécessitant donc une optimisation laborieuse de ces derniers. Cette optimisation constitue un obstacle majeur pour la translation de l’optophysiologie SERS vers le domaine clinique. Pour pallier à ce problème, plusieurs codes barre furent attribués par molécule et analyser, de manière analogue à de la reconnaissance faciale, par un réseau neuronal artificiel. L’optophysiologie SERS couplée à de l’intelligence artificielle a permis la détection de plus de sept métabolites extracellulaires associés au métabolisme de carbone, de l’azote et de l’énergie dans des cellules cancéreuses et contrôles. Les expériences présentées dans la thèse montrent le potentiel d’application de l’optophysiologie SERS pour la détection de métabolites sécrétés par des neurones, ou d’autres types de cellules présentant divers phénotypes, afin d’en étudier les fonctions biologiques normales ou celles associées à une pathologie. Ces nouvelles technologies mèneront certainement à des percées importantes dans différents domaines cliniques et scientifiques.To insure the signalling and regulation of their metabolism, living cells secrete a plethora of small and large molecule referred as chemical messengers. These messengers are generally secreted into the extracellular space, and diffuse until they reached a specific molecular target. The resulting biological recognition event between a chemical messenger and its dedicated molecular receptor induces a cascade of biochemical reactions, corresponding to the fundamental function of biological cells. However, a dysfunction associated to the secretion of these chemical messengers is linked to multiple clinical diseases. As an example, a change in the composition of neurotransmitters released during synaptic transmission in the brain is associated to Alzheimer disease, Parkinson disease, depression, addiction and schizophrenia. Thus, the analysis of the chemical messenger is of great clinical interest. However, modern analytical methods are ill suited to capture the fast and heterogeneous changes in chemical messengers during secretion events. New technologies allowing a sensitive and simultaneous monitoring of chemical messengers in a local and non-destructive way is needed. Plasmonic materials enable surface enhanced spectroscopies such as surface enhanced Raman scattering (SERS). With the molecular selectivity of SERS, and its high sensitivity, capable of detecting a single molecule, SERS sensors offer multiple advantages allowing the development of new technologies adapted for monitoring cellular secretion. The main objective of the thesis is the development of a novel SERS sensor capable of monitoring cellular secretion with both high sensitivity and selectivity. To achieve this goal, gold nanoparticles were electrostatically adsorbed onto a nanometric sized patch clamp glass capillary. The immobilization of the nanoparticles led to the generation of plasmonic nanosensors, which can be positioned in space while keeping a constant aggregation state, a key parameter in SERS. These nanosensors were then positioned near cells in vitro and SERS spectra were acquired in time. In order to analyze the complex optical response, novel chemometric approaches were developed. As a starting point, a unique barcode was extracted from each experimental SERS spectrum detected in time, and then compared with a spectral database of SERS barcode extracted from various standards. This approach enabled (1) identification of the molecule according to the barcode, and (2) a semi-quantitative analysis by counting the number of times the barcode, for each standard, was detected within a defined timescale. This chemometric method, referred as SERS optophysiology, was applied to monitor abundant metabolites in the extracellular environment near MDCKII cells in both basal and stimulated conditions. As this is a new method, SERS optophysiology required optimization of its hyper-parameters prior to the analysis, thus limiting its multiplex capability and adaptation to detect novel molecular targets. To improve the analytic performance of SERS optophysiology, we implemented a new algorithm to evaluate the barcodes. This new algorithm was applied to monitor neurotransmitters secreted by dopaminergic neurons from mice mesencephalon. Also, to visualize the neurons and correctly positioned the nanosensor near axonal varicosities, a hyphenated fluorescence-Raman microscope was built. With these tools, we successfully monitored the relative changes in concentration of five neurotransmitters in basal condition and throughout multiple stimulation cycles. Although improved, the analytical performances of SERS optophysiology depend mostly on the barcode data processing which thus required an extensive optimization to achieve good selectivity and sensitivity, limiting the translation of these technologies to other users. To overcome this limitation, multiple barcodes were thus associated to a single standard and analyzed with an artificial neural network, analogous to facial recognition. Machine learning driven SERS optophysiology enabled the detection of more than seven metabolites associated to the metabolism of carbon, nitrogen and energy of healthy and cancerous cell lines. The experiments presented in the thesis show the application potential of SERS optophysiology for the detection of secreted metabolites by neurons, or other type of cells exhibiting different phenotype, in order to investigate biological functions in a normal or disease state. These novel technologies thus hold a promising potential for significant discoveries in many fields of medicine and general science

Investigation in to the Use of Thermoplastic Nanochannels for Time of Flight (TOF) Detection of Nucleotide Monophosphates: Towards Single Molecule DNA Sequencing

Because of the unique properties that arise when the column size is comparable to either the length scale of electrostatic interactions or the size of the molecules being transported through them, nanochannel-based devices have garnered attention for many applications, especially nanoelectrophoresis. One essential application looking to exploit unique phenomena that occur at the nanoscale is Single Molecule Sequencing (SMS). SMS offers advantages over conventional ensemble-based sequencing platforms. Our proposed SMS device looks to identify nucleotides based on their molecular-dependent flight times as they migrate through a nanochannel, termed Time-of-Flight (ToF) detection. This research looked to understand the use of thermoplastic substrates for the fabrication of nanochannels to utilize in nanoelectrophoresis experiments for ToF detection. Differences in the migration properties of dNMPs under varying pH, buffer additives and buffer concentration to enhance the resolution of the separation and ultimately result in a high base calling accuracy were explored as well. Super Resolution Fluorescence data indicated non-uniform distributions of -COOH functional groups for both COC and PMMA thermoplastics with the degree of heterogeneity being dose dependent. In addition, COC demonstrated relative higher surface density of functional groups compared to PMMA for both UV/O3 and O2 plasma treatment. The spatial distribution of -COOH groups secured from super-resolution imaging were used to simulate non-uniform patterns of electroosmotic flow in thermoplastic nanochannels. Simulations were compared to single-particle tracking of fluorescent nanoparticles within thermoplastic nanoslits to demonstrate the effects of surface functional group heterogeneity on the electrokinetic transport process. Furthermore, results showed that increased norbornene content within COC led to the generation of more oxygen containing functionalities such as alcohols, ketones, aldehydes and carboxyl groups when activated with either UV/O3 or O2 plasma. Specifically, COC 6017 (~60% norbornene content) showed a significantly higher –COOH functional group density when compared to COC 6013 (~50% norbornene content) and COC 8007 (~35% norbornene content) following UV/O3 or O2 plasma activation. Furthermore, COC 6017 showed a smaller average RMS roughness (0.65 nm) when compared to COC 8007 (0.95 nm) following activation making this substrate especially suited for nanofluidic applications, which require smooth surfaces to minimize effects arising from dielectrophoretic trapping or non-specific adsorption. Although all COC substrates showed >90% transparency at wavelengths >475 nm, COC 6017 showed significantly less transparency at wavelengths below 475 nm following activation, making optical detection in this region difficult. Our data showed distinct physiochemical differences in activated COC that was dependent upon the ethylene/norbornene content of the thermoplastic and thus, careful selection of the particular COC grade must be considered for micro- and nanofluidics. Finally, we determined that nanoscale columns introduce unique surface interactions differences of the dNMPs allowing for resolutions ranging from 0.42-0.94 and changes in the pH that can further enhance resolutions up to 2.7. Furthermore, it was determined that low buffer concentrations resulting in EDL overlap decrease the resolution. In addition, nanoscale electrophoresis was performed on the sub-second time scale, resulting in highly efficient separations. Ultimately, our research shows great promise for the use of nanoelectrophoresis within thermoplastic columns for the separation of dNMPs among many other molecules, not achievable on the microscale.Doctor of Philosoph

Functional Gold Nanoparticles for Biomedical Applications

Abstract Subjects of the present dissertation are the synthesis, the functionalization and the characterization of colloidal gold nanoparticles. The employed nanoparticles consist of an inorganic Au core of approximately 5 nm diameter, which is stabilized by hydrophobic surface molecules. To transfer the nanoparticles to aqueous environments (an indispensable necessity for biomedical applications) they are coated with an amphiphilic polymer, which generates water solubility and moreover gives the ability for further functionalization. The physico-chemical properties of such nanoparticles are verified within different purposes: First, several fundamental intrinsic surface properties are quantified, including the establishment of pH titration as characterization tool. It is found that the carboxylic groups, responsible for the colloidal stabilization, partly have different properties (like their pKa) compared to free standing carboxylic acids. These findings are crucial for the colloidal stabilization of nanoparticles as well as for their further functionalization. Secondly, two species of fluorescently labeled nanoparticles, which differed in first order only in the net surface charge, are employed to study charge dependent interaction of nanoparticles with biological systems, including proteins as well as living cells. The main finding is, that a so called protein corona forms around nanoparticles, what has far-reaching impacts on cell internalization abilities. Moreover it is found that positively charged nanoparticles show a higher cell association as well as a higher toxicity. Thirdly, nanoparticles are modified towards sensing applications by surface functionalization with ion sensitive dyes. Positively charged nanoparticles are modified with a Cl- sensitive dye and negatively charged nanoparticles are modified with a Zn2+ sensitive dye. The goals of the dissertation can be synoptically depicted as: 1) Extension of the existing techniques for nanoparticle functionalization, particularly regarding new types of functional polymers. 2) A fundamental and comprehensive characterization of nanoparticles ranging from the verification of intrinsic, physico-chemical properties to biomedical applications

High Yield Production and Biofunctionalization of Plasma Polymerized Nanoparticles for Applications in Biomedicine

The advent of nanoparticle technology in the medical and pharmaceutical sectors offers significant potential in the quest for more effective healthcare solutions. Surface biofunctionalization, the engineering of nanoparticle surfaces for targeted biomedical applications, is critical for the development of personalized medicine in the delivery of diagnostic and therapeutic agents. However, the transition from laboratory research to commercial scale production faces substantial challenges in ensuring scalability of synthesis methods, consistent nanoparticle quality and addressing safety concerns related to toxicity. The commercial viability of nanoparticle-based medical products requires the precedence of translation into clinical settings warranting their integration into cellular environments while maintaining bioactivity. Traditional methods for nanoparticle synthesis and functionalization often fall short from achieving this aim due to inconsistencies in particle sizes, high production costs, reagent toxicity and subsequent complex wet-chemical processing. This thesis introduces plasma polymerization as a high-throughput method for producing surface- active polymeric nanoparticles, through a more environmentally friendly, dry synthesis process. Chapter 1 introduces polymer nanoparticles in nanomedicine, focusing on surface functionalization for improved diagnostics and therapeutics, and overcoming biological barriers for targeted delivery. Chapter 2 outlines methodologies. Chapter 3 discusses enhanced collection yield while maintaining properties for surface treatment, demonstrated through biofunctionalization of plasma polymerized nanoparticles (PPNs) with non-cytotoxic covalent bonds. Chapter 4 confirms this via in vitro studies. Chapter 5 studies long-term stability, showing bioactivity after a year. The final chapter details PPN synthesis, validating a continuum fluid model to enhance predictive accuracy