Scanning electron microscopy image representativeness: morphological data on nanoparticles.

A sample of a nanomaterial contains a distribution of nanoparticles of various shapes and/or sizes. A scanning electron microscopy image of such a sample often captures only a fragment of the morphological variety present in the sample. In order to quantitatively analyse the sample using scanning electron microscope digital images, and, in particular, to derive numerical representations of the sample morphology, image content has to be assessed. In this work, we present a framework for extracting morphological information contained in scanning electron microscopy images using computer vision algorithms, and for converting them into numerical particle descriptors. We explore the concept of image representativeness and provide a set of protocols for selecting optimal scanning electron microscopy images as well as determining the smallest representative image set for each of the morphological features. We demonstrate the practical aspects of our methodology by investigating tricalcium phosphate, Ca3 (PO4 )2 , and calcium hydroxyphosphate, Ca5 (PO4 )3 (OH), both naturally occurring minerals with a wide range of biomedical applications

Particulate airborne impurities

The cumulative effects of air pollutants are of principal concern in research on environmental protection in Sweden. Post-industrial society has imposed many limits on emitted air pollutants, yet the number of reports on the negative effects from them is increasing, largely due to human activity in the form of industrial emissions and increased traffic flows. Rising concerns over the health effects from airborne particulate matter (PM) stem from in vitro, in vivo, and cohort studies revealing effects of mostly negative nature. Full insight into the health effects from PM can only be achieved through practical investigation of the mode of toxicity from distinct types of particles and requires techniques for their identification, monitoring, and the production of model fractions for health studies. To this effect, comprehensive collection and chemical analysis of particulates at the origin of emission was performed in order to provide clearer insight into the nature of the particulates at exposure and add detail to aid risk assessment. Methods of capturing particles and analyzing their chemical nature were devised using scanning electron microscopy coupled with energy dispersive spectroscopy (SEM-EDS). Furthermore, taking the approach of in vitro cytotoxicity testing, nanoparticles of types typical to automotive emissions, were synthesized and extensively characterized using SEM-EDS, X-ray diffraction (XRD), transmission electron microscopy (TEM),dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). The produced model magnetite and palladium nanoparticles were found to induce toxicity in human pulmonary epithelial cells (A549 and PBEC) as well as impact severely on immunological and renal cells (221 B- and 293T-cells) in a dose-dependent manner

Functionalized silica nanostructures for biosensing applications

This work covers both two dimensional (2D) and three dimensional (3D) silica-based nanostructures for use in biomedical sensing applications. The first section of this study discusses the formation of 2D nanostructured surface plasmon resonance (SPR)-based biosensor substrates. The surface of these biosensors was nanostructured by adding sacrificial star polymers or block copolymers to a silicate precursor solution. Subsequent vitrification resulted in two distinct morphological patterns: random and ordered porosity. Amino groups on the surface of the biosensors enabled the installation of analyte receptors and antifouling agents such as oligo (ethylene oxide). The second section discusses the development of 3D core-shell silica nanoparticles (SNPs). For this work, star polymers were generated to provide hydrophobic interiors capable of sequestering large hydrophobic porphyrinoid dyes and hydrophilic exteriors capable of templating the growth of silica shells. The diameter of the SNPs (25-100 nm) varied depending on reaction time, template size, and reagent concentration. The shell thickness was also controlled in order to either release or retain the hydrophobic dyes. The SNPs were surface-functionalized with biocompatible stealth materials such as poly (ethylene oxide) to generate non-toxic, water-soluble nanoparticles for the in vivo delivery of various hydrophobic imaging and therapeutic materials

Electrochemically engineered anodic alumina Nanotubes: physico-chemical properties and Applications

Des del seu descobriment, l'alúmina anòdica porosa s’ha utilitzat com a recobriment protector. El descobriment de la seva estructura porosa va animar els investigadors a desenvolupar nous mètodes de fabricació d'alúmina, obtenint així geometries complexes de propietats diverses. En aquesta tesi es desenvolupen nanotubs d'alúmina anòdica (AANTs) mitjançant un procés d’anodització que es coneix com anodització per polsos. El procés consisteix a entrellaçar polsos de corrent de baixa (~6 mA/cm2) i alta (~290-390 mA/cm2) densitat. Un flux de corrent prou alt produeix un estrenyiment vertical dels porus i unions entre cel·les més febles. L'atac electroquímic selectiu i la sonicació en aigua de l'estructura resultant permeten produir col·loides de nanotubs. El primer objectiu d'aquesta tesi és una anàlisi exhaustiva del procés per comprendre millor el mecanisme de formació dels AANTs i relacionar les condicions d’anodització amb la seva geometria resultant. El segon objectiu és avaluar i optimitzar el seu postprocessat, investigant nous mètodes d'alteració de les seves propietats fisicoquímiques. L'últim objectiu és dissenyar i fabricar nanotubs i proposar les seves aplicacions. Aquest treball investiga l'evolució del perfil de l'alúmina en funció dels paràmetres d’anodització. A més, el corrent i el potencial del procés s'associen amb la geometria i les propietats dels nanotubs obtinguts: longitud, diàmetre intern i extern, potencial Z i tamany. En resum, un corrent més alt condueix a nanotubs més llargs i estrets amb menor càrrega superficial. S'avaluen i optimitzen les condicions de sonicació. Es demostra que el recuit a alta temperatura dels nanotubs té un impacte en la seva estructura cristal·lina i composició elemental. Posteriorment, els nanotubs es decoren electrostàticament amb nanopartícules magnètiques i es modifica el seu interior amb una proteïna marcada amb fluoròfor. Aquests col·loides magnètics han demostrat ser útils per a la detecció de la catepsina B, el que demostra la seva utilitat com a sensors.La anodización del aluminio tiene casi un siglo de historia. La alúmina anódica se utilizó inicialmente como recubrimiento protector, pero el desarrollo de la microscopía electrónica reveló la morfología porosa de este óxido. Este descubrimiento animó a los investigadores a desarrollar nuevos métodos de fabricación de la alúmina porosa, obteniendo así geometrías complejas con diversas propiedades. En esta tesis se desarrollan nanotubos de alúmina anódica (AANTs) a través de un proceso de anodización que se conoce como anodización por pulsos. El proceso consiste en entrelazar pulsos de corriente de baja (~ 6 mA / cm2) y alta (~ 290-390 mA / cm2) densidad. Un flujo de corriente suficientemente alto afecta a la formación de la estructura, resultando en un estrechamiento vertical de los poros y uniones entre celdas más débiles. El ataque electroquímico selectivo y la sonicación en agua de la estructura resultante permiten producir coloides de nanotubos. El primer objetivo de esta tesis es un análisis exhaustivo del proceso para comprender mejor el mecanismo de formación de los AANTs y conectar con precisión las condiciones de anodización con la geometría resultante de la estructura. El segundo objetivo es evaluar y optimizar su posprocesado, investigando nuevas posibilidades de alterar las propiedades fisicoquímicas de los AANT. El último objetivo es diseñar y fabricar nanotubos funcionales y proponer sus aplicaciones. Este trabajo investiga la evolución del perfil de anodización en función de las condiciones del proceso de anodización. Además, la corriente y el potencial del proceso se asocian con la geometría y las propiedades de los nanotubos obtenidos: longitud, diámetro interno y externo, potencial Z y dispersión de tamaño. En resumen, una corriente más alta conduce a nanotubos más largos y estrechos con una carga superficial más baja. Se evalúan las condiciones de sonicación proponiendo un conjunto de parámetros más óptimo. Se demuestra que el recocido a alta temperatura de los nanotubos tiene un impacto en su estructura cristalina y composición elemental: el aumento de temperatura produce una fracción cristalina más alta y disminuye su contenido de azufre. Posteriormente, los nanotubos se decoran electrostáticamente con nanopartículas de maghemita y se modifica su interior con una proteína marcada conMost of the time since its discovery, nanoporous anodic alumina was used as a protective coating. The intrinsic property revealed by the electron microscope – porosity – encouraged researchers to investigate new methods of porous alumina fabrication, obtaining complex geometries with various properties. In this thesis, anodic alumina nanotubes (AANTs) are developed through a carefully adjusted anodization process defined as pulse anodization. The process consists of interlacing current pulses of low (~6 mA/cm2) and high (~290-390 mA/cm2) density. Sufficiently high current flow affects the formation of the structure, resulting in vertical pore narrowings and weaker cell junctions. Selective acid etching and sonication in water enables to yield colloids of nanotubes. First aim of this thesis is a thorough analysis of the process to better understand the formation mechanism of AANTs and precisely connect anodization conditions with the resultant geometry of the structure. Second goal is to evaluate and optimize post-processing investigating further possibilities to alter physio-chemical properties of AANTs. Last objective is to design and fabricate functional nanotubes and propose their applications. This work reports the evolution of the anodization profile depending on the process conditions. Further, current and potential of the process are associated with the geometry and the properties of the obtained nanotubes: length, inner and outer diameter, z-potential and size dispersity. In brief, higher current leads to longer and narrower nanotubes with lower surface charge. Sonication conditions are evaluated leading to the proposal of a more optimal set of parameters. Annealing of the nanotubes is demonstrated to impact on their crystalline structure and elemental composition: temperature increase leads to higher crystalline fraction and decrease their sulfur content. Nanotubes are later electrostatically-decorated with maghemite nanoparticles and modified inside with a fluorophore labelled protein. These magnetically responsive colloids demonstrate stimuli-responsive detection of cathepsin B, supporting its utility as a sensor

Chiral Metafilms and Surface Enhanced Raman Scattering For Enantiomeric Discrimination of Helicoid Nanoparticles

Chiral nanophotonic platforms provide a means of creating near fields with both enhanced asymmetric properties and intensities. They can be exploited for optical measurements that allow enantiomeric discrimination at detection levels greater than 6 orders of magnitude than is achieved with conventional chirally sensitive spectroscopic methods based on circularly polarized light. The optimal approach for exploiting nanophotonic platforms for chiral detection would be to use spectroscopic methods that provide a local probe of changes in the near field environment induced by the presence of chiral species. Here we show that surface enhanced Raman spectroscopy (SERS) is such a local probe of the near field environment. We have used it to achieve enantiomeric discrimination of chiral helicoid nanoparticles deposited on left and right-handed enantiomorphs of a chiral metafilm. Hotter electromagnetic hotspots are created for matched combinations of helicoid and metafilms (left-left and right-right), while mismatched combinations leads to significantly cooler electromagnetic hotspots. This large enantiomeric dependency on hotspot intensity is readily detected using SERS with the aid of an achiral Raman reporter molecule. In effect we have used SERS to distinguish between the different EM environments of the plasmonic diastereomers produced by mixing chiral nanoparticles and metafilms. The work demonstrates that by combining chiral nanophotonic platforms with established SERS strategies new avenues in ultrasensitive chiral detection can be opened

Vertical distribution of inorganic nanoparticles in a Norwegian fjord

Due to the analytical challenges of detecting and quantifying nanoparticles in seawater, the data on distributions of NPs in the marine environment is limited to qualitative studies or by ensemble measurements subject to various analytical artifacts. Single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) allows determination of individual inorganic NPs at environmentally relevant concentrations, yet only few studies have been conducted on selected elements in surface sea water. Here, a sequential multi-element screening method was developed and implemented to provide a first survey of the horizontal and vertical distributions of inorganic nanoparticles and trace elements in a pristine Norwegian fjord prospect for submarine tailings deposition. Statistical control of false-positive detections while minimizing the size detection limit was ensured using a novel raw signal processing. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) gave confirmative and qualitative information regarding particle morphology and composition. Following SP-ICP-MS screening for particles of 16 elements, particulate Al, Fe, Mn, Pb, Si and Ti were found and determined to mass concentrations in ng/L of 1–399, 1–412, below limit of detection (<LOD) - 269, <LOD - 1, <LOD - 1981 and <LOD - 127 ng/L with particle number concentrations up to 108 particles per liter. Total metals concentrations were at least an order of magnitude higher, at concentrations in μg/L of 1–12 for Al, 2–13 for Fe, 0.3–11 Mn, 0.02–0.5 for Pb, 46 to 318 Si and 0.04–0.4 for Ti. A strong depth dependence was observed for both trace elements and particles with concentrations increasing with depth. Our results provide a baseline for the fjord and new data on environmental levels of both total metals and metal containing nanoparticles including the vertical and horizontal distribution of natural nanoparticlesVertical distribution of inorganic nanoparticles in a Norwegian fjordpublishedVersio

Towards a review of the EC Recommendation for a definition of the term "nanomaterial"; Part 1: Compilation of information concerning the experience with the definition

In October 2011 the European Commission (EC) published a Recommendation on the definition of nanomaterial (2011/696/EU). The purpose of this definition is to enable determination when a material should be considered a nanomaterial for regulatory purposes in the European Union. In view of the upcoming review of the current EC Definition of the term 'nanomaterial' and noting the need expressed by the EC Environment Directorate General and other Commission services for a set of scientifically sound reports as the basis for this review, the EC Joint Research Centre (JRC) prepares three consecutive reports, of which this is the first. This Report 1 compiles information concerning the experience with the definition regarding scientific-technical issues that should be considered when reviewing the current EC definition of nanomaterial. Based on this report and the feedback received, JRC will write a second, follow-up report. In this Report 2 the JRC will provide a detailed assessment of the scientific-technical issues compiled in Report 1, in relation to the objective of reviewing the current EC nanomaterial definition.JRC.I.4-Nanobioscience

Novel nanoparticle formulations for antimicrobial action

Colloidal particles are being extensively studied in various antimicrobial applications due to their small size, enormous surface area to volume ratio and ability to exhibit a wide spectrum of antibacterial, antifungal, antialgal and antiviral action. This thesis aims to develop novel nanoparticle formulations for antimicrobial action. The present work focuses on various nanoparticles (NPs) of inorganic materials and discusses some of the methods for their preparation as well as mechanisms of their antimicrobial action. The antimicrobial applications of metal oxide nanoparticles (zinc oxide and copper oxide) and metal hydroxide nanoparticles such as magnesium hydroxide were studied. Recent advances in the functionalization of nanoparticles and their potential antimicrobial applications were also studied as a viable alternative of conventional antibiotics and antiseptic agents which can help to tackle antimicrobial resistance.The synthesis and characterisation of a range of surface modified zinc oxide (ZnONPs, Chapter 3 and 4), magnesium hydroxide Mg(OH)2NPs (Chapter 5 and 6) and copper oxide (CuONPs, Chapter 7 and 8) have been described including particle size distribution, crystallite size, zeta potential, isoelectric point, X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscopy (SEM), etc. The antibacterial, anti-algal and anti-yeast activity of the modified nanoparticles on microalgae (C. reinhardtii), yeast (S. cerevisiae) and Escherichia coli (E.coli) were explored. The viability of these cells was evaluated for various concentrations and exposure times with nanoparticles. It was discovered that the antimicrobial activity of uncoated nanoparticles on the viability of C. reinhardtii occurred at considerably lower particle concentrations than for S. cerevisiae and E.coli. The results indicate that the antimicrobial activity of polyelectrolyte-coated nanoparticles alternates with their surface charge. The anionic nanoparticles (ZnONPs/PSS, ZnONPs/ZnS, ZnONPs/SiO2, CuONPs/PSS and Mg(OH)2NPs/PSS) have much lower antibacterial activity than the cationic ones (NPs/PSS/PAH and uncoated NPs). These findings have been explained by the lower adhesion of the anionic nanoparticles to the cell wall because of electrostatic repulsion and the enhanced particle-cell adhesion due to electrostatic attraction in the case of cationic nanoparticles. The results can potentially be applied to control the cytotoxicity and the antimicrobial activity of other inorganic nanoparticles.A novel type of antimicrobial formulation of CuONPs has been developed and tested. This has been achieved by functionalizing CuONPs with (3-glycidyloxypropyl)- trimethoxysilane (GLYMO) and subsequent covalent coupling of 4- hydroxyphenylboronic acid (4-HPBA). As the boronic acid (BA) groups on the surface of CuONPs/GLYMO/4-HPBA can form reversible covalent bonds with the diol groups of glycoproteins on the bacterial cell surface, they can strongly bind to the cells walls resulting in a very strong enhancement of their antibacterial, anti-algal and anti-yeast action which is not based on electrostatic adhesion. This work (Chapter 8) demonstrates that the CuONPs with boronic acid surface functionality are far superior antibacterial agents compared to bare CuONPs. The results showed that the antibacterial, anti-algal and anti-yeast impact of the 4-HPBA-functionalized CuONPs on Rhodococcus rhodochrous (R. rhodochrous), E.coli, C. reinhardtii and S. cerevisiae is one order of magnitude higher than that of bare CuONPs or CuONPs/GLYMO. It was also observed a marked increase of the 4-HPBA-functionalized CuONPs antibacterial action on these microorganisms at shorter incubation times compared with the bare CuONPs at the same conditions. Significantly, the results show that the cytotoxicity of CuONPs functionalized with 4-HPBA as an outer layer can be controlled by the concentration of glucose in the media, and that the effect is reversible as glucose competes with the sugar residues on the bacterial cell walls for the BA-groups on the CuONPs. The experiments with human keratinocyte cell line exposure to CuONPs/GLYMO/4-HPBA indicated lack of measurable cytotoxicity at particle concentrations which are effective as an antibacterial agent for both R. rhodochrous, E. coli, C. reinhardtii and S. cerevisiae. This suggests that formulations of CuONPs/GLYMO/4-HPBA can be used to drastically reduce the overall CuO concentration in antimicrobial formulations while strongly increasing their efficiency.The role of surface roughness in the antimicrobial activity of oxide nanoparticles has been studied (Chapter 9). This has been achieved by comparing the antimicrobial action of non-porous silica nanoparticles (SiO2NPs) with smooth surface and mesoporous surfacerough SiO2NPs, both functionalized with GLYMO and 4-HPBA. Surface-rough mesoporous silica nanoparticles (‘ghost’ SiO2NPs) have been fabricated by using composite mesoporous copper oxide nanoparticles (‘host’ CuONPs) as templates which allowed the SiO2NPs to copy their surface morphology. It was demonstrated that the functionalized ‘ghost’ SiO2NPs with GLYMO and 4-HPBA (SiO2NPs/GLYMO/4-HPBA) show a very significant antibacterial effect compared to smooth SiO2NPs of the same surface coating and particle size. This was attributed to the ‘ghost’ SiO2NPs surface morphology which mimics to certain extent the surface of the original CuONPs used as templates for their preparation. It can be envisaged that the ‘ghost’ SiO2NPs effectively acquire some of the antibacterial properties from the ‘host’ CuONPs, with the same functionality, despite being completely free of copper. Antibacterial tests showed that the ‘ghost’ SiO2NPs/GLYMO/4-HPBA have much higher antibacterial action than the nonfunctionalized ‘ghost’ SiO2NPs or GLYMO functionalized ‘ghost’ SiO2NPs for R. rhodochrous. The results indicate that the combination of rough surface morphology and strong adhesion of the particle surface to the bacteria can make even benign material as silica act as a strong antimicrobial

Development of a method for bioanalysis of a nanomaterial in biological matrices

Molecular imaging techniques have become an essential tool for cancer diagnosis. They have the potential to detect cancer at early stages and thus, they can change the outcome of the disease and patient prognosis. Magnetic resonance imaging (MRI) has arisen as one of the most promising imaging methods used for the screening of soft tumor tissues such as breast cancer. However, the specificity of MRI remains poor leading to misdiagnosis and many false positive findings. Thus, a wide range of new contrast agents (CAs) are being developed in order to enhance image resolution and safety in cancer diagnosis. For new drugs with an intended clinical use, it is necessary a deep insight into the viability of the molecule prior to clinical trials. Some in vitro analysis and in vivo animal studies are carried out in order to characterize the physicochemical properties of the compound and its toxicity. Problems to find the right methodology to detect and assess identity of nanoparticles after being injected into the blood have been previously found. In this project, a combined method to extract and characterize dummy particles, mimicking nanoparticles to be used as CAs, was developed. These particles consist of a polymeric core, which contains a metal ion inside, together with a coating attached to the surface. A new approach for characterization was investigated by using three analytical techniques: gel permeation chromatography (GPC) to extract and separate the nanoparticles by size, enzyme-linked immunosorbent assay (ELISA) to analyze the coating and inductively coupled plasma optical emission spectroscopy (ICP-OES) to analyze the core and metal ion. Different biological matrices, mainly serum and urine, were tested to prove identity. Results showed an efficient extraction of the material with a high rate of recovery by GPC. Moreover, the accuracy and sensitivity of ELISA and ICP-OES in detecting the coating and composition of nanoparticles respectively was proved. More importantly, identity of the extracted material in the biological matrices was demonstrated. Future studies are required to scale up the method and further test blood samples from in vivo trials to completely implement the method. However, here the first steps towards the validation of the method were performed which can help to understand changes in nanoparticles upon exposure to biological fluids in the body.Tracing nanoparticles in biological matrices Cancer includes a heterogeneous group of diseases that arises due to an uncontrolled proliferation of cells which are able to invade normal tissues. Magnetic resonance imaging (MRI) has been widely used to detect cancer in its earliest stage. However, there is a need for new contrast agents (CAs) with higher sensitivity. Consequently, cancer nanotechnology has emerged as a new potential field which includes the design of material in a scale range from 1-100 nanometers with clinical use for cancer therapy and diagnosis. Nanomaterials include a wide range of particles used as versatile molecular devices like vectors for drug delivery or CAs to enhance contrast images from tumor tissues. Spago Nanomedical AB in Lund is studying a potential nanomaterial-based contrast agent to be used in MRI. The main structure of the nanoparticle consists of a polymeric central core, which contains a metal ion inside, together with a coating attached to the surface (Figure 1). This coating is made of polyethylene glycol (PEG) chains which form a passive surface that reduces aggregation and undesired interactions in the body. These particles can be delivered into the tumor tissue through passive targeting. This strategy can be used because during their growth, cancer cells create new but defective blood vessels. Thus, tumor vasculature has an enhanced permeability and lack of an efficient lymphatic drainage which gives rise to the so called enhanced permeability and retention (EPR) effect. This effect allows the nanoparticles to accumulate selectively in the tumor and remain there for a longer time compared to a normal tissue. Do nanoparticles look the same after being injected into the body? In this project, a dummy particle mimicking a nanoparticle-based contrast agent was further investigated to set up a combined bioanalysis method to extract and characterize the nanomaterial from relevant biological matrices (e.g urine, serum). The final goal was to demonstrate that the identity of the particles (the main physicochemical properties) remain unaffected after being injected into the blood of animals during in vivo trials. Problems can arise if the content of the nanoparticles is released to the biological fluids or if particles aggregate and lodge somewhere in the body. The nanoparticles were isolated and separated by size using gel permeation chromatography (GPC). The resulting fractions were analyzed in terms of composition and coating using inductively coupled plasma optical emission spectroscopy (ICP-OES) and enzyme-linked immunosorbent assay (ELISA) respectively. The results proved the presence of nanomaterial in the biological matrices and showed a successful separation of the nanoparticles by size with high rate of recovery. Furthermore, it was possible to analyze the core and coating components of the extracted material in an accurate and efficient way. This study shows the importance of characterizing the nanomaterial and proves its identity before going into clinical trials. Advisor: Dr. Sania Bäckström Master´s Degree Project in Molecular Biology, 45 credits, 2015 Department of Biology, Lund Universit