Search

Nonlinear micro-electro-mechanical systems (MEMS) resonators open new opportunities in sensing and signal manipulation compared to their linear counterparts by enabling frequency tuning and increased bandwidth. Here, we design, fabricate and study drumhead resonators exhibiting strongly nonlinear dynamics and develop a reduced order model (ROM) to capture their response accurately. The resonators undergo electrostatically-mediated thermoelastic buckling which tunes their natural frequency from 4.7 to 11.3 MHz, a factor of 2.4x tunability. Moreover, the imposed buckling switches the nonlinearity of the resonators between purely stiffening, purely softening, and even softening-to-stiffening. Accessing these exotic dynamics requires precise control of the temperature and the DC electrostatic forces near the resonator's critical-buckling point. To explain the observed tunability, we develop a one-dimensional physics-based ROM that predicts the linear and nonlinear response of the fundamental bending mode of these drumhead resonators. The ROM captures the dynamic effects of the internal stresses resulting from three sources: The residual stresses from the fabrication process, the mismatch in thermal expansion between the constituent layers, and lastly, the applied electrostatic forces. The ROM replicates the observed tunability of linear (within 5.5% error) and nonlinear responses even near the states of critical buckling. These remarkable nonlinear and large tunability of the natural frequency are valuable features for on-chip acoustic devices in broad applications such as signal manipulation, filtering, and MEMS waveguides.

Two-dimensional (2D) materials and heterostructures are promising candidates for nanoelectronics. However, the quality of material interfaces often limits the performance of electronic devices made from atomically thick 2D materials and heterostructures. Atomic force microscopy (AFM) tip-based cleaning is a reliable technique to remove interface contaminants and flatten heterostructures. Here, we demonstrate AFM tip-based cleaning applied to hBN-encapsulated monolayer MoS2 transistors, which results in electrical performance improvements of the devices. To investigate the impact of cleaning on device performance, we compared the characteristics of as-transferred heterostructures and transistors before and after tip-based cleaning using photoluminescence (PL) and electronic measurements. The PL linewidth of monolayer MoS2 decreased from 84 meV before cleaning to 71 meV after cleaning. The extrinsic mobility of monolayer MoS2 field-effect transistors increased from 21 cm2/Vs before cleaning to 38 cm2/Vs after cleaning. Using the results from AFM topography, photoluminescence, and back-gated field-effect measurements, we infer that tip-based cleaning enhances the mobility of hBN-encapsulated monolayer MoS2 by reducing interface disorder. Finally, we fabricate a MoS2 field-effect transistor (FET) from a tip-cleaned heterostructure and achieved a device mobility of 73 cm2/Vs. The results of this work could be used to improve the electrical performance of heterostructure devices and other types of mechanically assembled van der Waals heterostructures.

Objectives: To investigate which opinions among dentists are associated with level of technology use, when characteristics of the dentist and dental practice, as well as motivating work aspects are taken into account. Methods: A total of 1000 general dental practitioners in the Netherlands received a questionnaire on digital technologies they use, opinions on using technologies and related motivating work aspects. Questions were derived from expert interviews, the Dentists’ Experienced Job Resources Scale and literature on technology implementation. Technology use was measured as the number of technologies used, and divided into three technology user groups: low (using 0-4 technologies, mostly administrative and radiographic technologies), intermediate (using 5-7, more varied technologies) and high technology users (using 8-12, including more innovative diagnostic technologies). Opinions on technology use and motivating work aspects were analysed using principal components analysis (PCA) and exploratory factor analysis. Scores on all components and factors were calculated for each respondent by computing the mean of all valid responses on the underlying items. Differences in these scale scores on opinions among the technology user groups were assessed using one-way analysis of variance and Kruskal–Wallis tests. A multiple linear regression analysis assessed the association of scale scores about opinions on technology use with the sum of technologies used, taking into account motivating work aspects and characteristics of the dentist and dental practice. Results: The response rate was 31%. Dentists who were high technology users perceived technologies as yielding more improvements in quality of care, adding more value to the dental practice and being easier to use, than low technology users. High technology users thought technologies added more value to their work and they reported higher skills and resources. They also focused more on technologies and thought these are more ready to use than low technology users. High technology users derived more motivation from “Immediate results” and “Craftsmanship” than low technology users. Personal and practice characteristics, motivating work aspects, and the opinion scales “Focus” and “Added value to dentist” explained 50% of the variance in the number of technologies a dentist uses. Conclusion: Opinions on digital technologies among dentists and motivating work aspects vary with level of technology use. Being more focused on technologies and perceiving a higher added value from using them are associated with using more digital dental technologies, when taking into account motivating work aspects and characteristics of the dentist and dental practice.

Introduction: There is a growing body of evidence for associations between oral and other chronic non-communicable diseases. Early recognition and diagnosis can be important in treating these diseases and better integration of primary and dental care could improve patient care. With the Dent@Prevent project, we aim to develop an electronic decision support system (DSS) and interactive mobile application to promote better collaboration between general practitioners (GPs) and dentists. Methods: A mixed-methods approach, including a systematic literature review, routine data analysis, focus groups with GPs and dentists and a Delphi-method with GPs, dentists and patients, is used to acquire information to develop the interactive mobile application that assesses patient-reported measures and the DSS that supports GP and dentist decision-making in an evidence-oriented way. Both the mobile application and DSS will be piloted to validate the use of these products. Results Findings: from the systematic literature review yielded a primary focus for the mobile application and DSS on links between diabetes and periodontitis. From the focus groups with dentists and GPs it appeared that GPs are generally less aware of interactions between oral and non-communicable diseases than dentists. Furthermore, several intervention points and integration of care processes were discussed as a basis for the DSS. The developed mobile application includes patient-reported measures for diabetes and periodontitis risks, as well as measures of patient experiences and perceptions towards the integration of care. The DSS is being designed to support the GP and dentist to recognize respectively an increased risk for periodontitis in their diabetes patients and an increased risk for diabetes in their patients with periodontitis, and to care for their patients accordingly. Discussions and conclusions: Electronic decision support systems for integration of primary and dental care might provide innovative and novel possibilities to improve integration of care through better informed decision making, including at the health policy level. Lessons learned: Clinicians may be unaware of interactions between oral and non-communicable diseases, which may reduce the chance for early recognition of disease in both sectors of care. Limitations: The Delphi-method was used to determine the content of the mobile application with regards to measures of patient experiences and perceptions. It was performed with an anonymized panel including dentists, GPs and patients. Because of this anonymity, the characteristics such as gender and age of the panel are unknown. Furthermore, because of limited scientific evidence on integrated care for diabetes patients with periodontitis, several development steps of the DSS depend on expert opinions from clinicians which may not be generally representative. Finally, the DSS we are developing is a pilot version but this seems a sensible approach towards stepwise creation of the DSS. Suggestions for future research: The mobile application and DSS have to be tested and validated in real-life settings before implementation in actual practice. Once the mobile application and DSS have been validated, the system can be evaluated to assess the efficiency of the intervention to promote integrated health under day-to-day practice conditions.

Introduction: Several chronic non-communicable diseases share common risk factors.Integration of care is important to ensure the best quality and efficiency of care for patients with multiple conditions. Patients’ perceptions are important for assessing the integration of care. For example,oral and general health are closely related. However, oral and general healthcare often take place in a disintegrated manner. This study aimed to develop a short and simple survey to assess integration of oral and general healthcare through patient perceptions. In addition, the survey was intended to be developed such that it can easily be implemented as an add-on to other surveys. Methods: A literature search was performed to develop a conceptual framework and set of questions for the Intersectoral Care Reported by Patients (ICRP) survey. Thereafter, a modified Delphi-method was used to further elaborate the survey. A panel consisting of seven international experts assessed the survey in three rounds. The experts were based in the United States, Germany, and the Netherlands and had different professional backgrounds including general practice and dentistry. During two interactive discussion rounds and one online round of voting, the framework and the questions were assessed, adapted, and prioritized by the experts. During the online round, the experts were asked to rate the necessity of each question and rank the questions based on importance for inclusion in the ICRP survey. Results: The co-creative development process yielded thirteen questions for inclusion in the ICRP survey. These questions focus on five domains: patients’ wishes, expectations and awareness regarding communication between healthcare providers; patients’ perception of healthcare providers’ knowledge; healthcare provider-patient communication about health status; utilization of healthcare; and self-rated health. Discussion and conclusions: We developed a short, easy-to-understand survey to assess integration of oral and general healthcare as perceived by patients. The survey provides opportunities for usage in research and as a tool in feedback systems for healthcare providers. Lessons learned: During the first group discussion, there were many suggestions for questions, including many differently formatted questions for the same concept. By asking the experts to rate the necessity and rank the questions during the online round, we were able to determine which questions were deemed most apt and relevant for inclusion in the ICRP survey. Limitations: The panel included seven experts, although not all experts were able to participate in all rounds of group conversations. This was solved through individual discussion with some of the experts. Although patients were not involved in the development of the survey, they will soon be involved in usability and understandability testing and piloting of the survey. Suggestions for future research: The survey’s understandability will be assessed by patients. Furthermore, the survey will be tested for usability in real-life practice settings. In order to compare different countries, the survey will be translated to other languages such as Dutch and German.

Background Antimicrobial resistance (AMR) is high on the UK public health policy agenda, and poses challenges to patient safety and the provision of health services. Widespread prescribing of antibiotics is thought to increase AMR, and mostly takes place in primary medical care. However, prescribing rates vary substantially between general practices. The aim of this study was to understand contextual factors related to general practitioners’ (GPs) antibiotic prescribing behaviour in low, high, and around the mean (medium) prescribing primary care practices. Methods Qualitative semi-structured interviews were conducted with 41 GPs working in North-West England. Participants were purposively sampled from practices with low, medium, and high antibiotic prescribing rates adjusted for the number and characteristics of patients registered in a practice. The interviews were analysed thematically. Results This study found that optimizing antibiotic prescribing creates tensions for GPs, particularly in doctor-patient communication during a consultation. GPs balanced patient expectations and their own decision-making in their communication. When not prescribing antibiotics, GPs reported the need for supportive mechanisms, such as regular practice meetings, within the practice, and in the wider healthcare system (e.g. longer consultation times). In low prescribing practices, GPs reported that increasing dialogue with colleagues, having consistent patterns of prescribing within the practice, supportive practice policies, and enough resources such as consultation time were important supports when not prescribing antibiotics. Conclusions Insight into GPs’ negotiations with patient and public health demands, and consistent and supportive practice-level policies can help support prudent antibiotic prescribing among primary care practices.

Nanoparticle in vitro toxicity studies often report contradictory results with one main reason being insufficient material characterization. In particular the characterization of nanoparticles in biological media remains challenging. Our aim was to provide robust protocols for two of the most commonly applied techniques for particle sizing, i.e. dynamic light scattering (DLS) and differential centrifugal sedimentation (DCS) that should be readily applicable also for users not specialized in nanoparticle physico-chemical characterization. A large number of participants (40, although not all participated in all rounds) were recruited for a series of inter-laboratory comparison (ILC) studies covering many different instrument types, commercial and custom-built, as another possible source of variation. ILCs were organized in a consecutive manner starting with dispersions in water employing well-characterized near-spherical silica nanoparticles (nominal 19 nm and 100 nm diameter) and two types of functionalized spherical polystyrene nanoparticles (nominal 50 nm diameter). At first each laboratory used their in-house established procedures. In particular for the 19 nm silica particles, the reproducibility of the methods was unacceptably high (reported results were between 10 nm and 50 nm). When comparing the results of the first ILC round it was observed that the DCS methods performed significantly worse than the DLS methods, thus emphasizing the need for standard operating procedures (SOPs). SOPs have been developed by four expert laboratories but were tested for robustness by a larger number of independent users in a second ILC (11 for DLS and 4 for DCS). In a similar approach another SOP for complex biological fluids, i.e. cell culture medium containing serum was developed, again confirmed via an ILC with 8 participating laboratories. Our study confirms that well-established and fit-for-purpose SOPs are indispensable for obtaining reliable and comparable particle size data. Our results also show that these SOPs must be optimized with respect to the intended measurement system (e.g. particle size technique, type of dispersant) and that they must be sufficiently detailed (e.g. avoiding ambiguity regarding measurand definition, etc.). SOPs may be developed by a small number of expert laboratories but for their widespread applicability they need to be verified by a larger number of laboratories., This work has been supported by the EU FP7 Capacities project QualityNano (grant no. INFRA-2010-262163).

The original version of this Article contained an error in the second sentence of the second paragraph of the ‘Electrical properties of fluorinated graphene contacts’ section of the Results, which incorrectly read ‘The mobility was calculated by the Drude model, μ = ne/σ where μ, n, e, and σ are the carrier mobility, carrier density, electron charge, and sheet conductivity, respectively’. The correct version states ‘μ = σ/ne ’ in place of ‘μ = ne/σ ’. This has been corrected in both the PDF and HTML versions of the Article.

Ridged, orthorhombic two-dimensional atomic crystals with a bulk {\em Pnma} structure such as black phosphorus and monochalcogenide monolayers are an exciting and novel material platform for a host of applications. Key to their crystallinity, monolayers of these materials have a four-fold degenerate structural ground state, and a single energy scale $E_C$ (representing the elastic energy required to switch the longer lattice vector along the $x-$ or $y-$direction) determines how disordered these monolayers are at finite temperature. Disorder arises when nearest neighboring atoms become gently reassigned as the system is thermally excited beyond a critical temperature $T_c$ that is proportional to $E_C/k_B$. $E_C$ is tunable by chemical composition and it leads to a classification of these materials into two categories: (i) Those for which $E_C\ge k_BT_m$, and (ii) those having $k_BT_m>E_C\ge 0$, where $T_m$ is a given material's melting temperature. Black phosphorus and SiS monolayers belong to category (i): these materials do not display an intermediate order-disorder transition and melt directly. All other monochalcogenide monolayers with $E_C>0$ belonging to class (ii) will undergo a two-dimensional transition prior to melting. $E_C/k_B$ is slightly larger than room temperature for GeS and GeSe, and smaller than 300 K for SnS and SnSe monolayers, so that these materials transition near room temperature. The onset of this generic atomistic phenomena is captured by a planar Potts model up to the order-disorder transition. The order-disorder phase transition in two dimensions described here is at the origin of the {\em Cmcm} phase being discussed within the context of bulk layered SnSe., Accepted on 01/11/2016. This document is the Submitted Manuscript version of a Published Work that appeared in final form in Nano Letters, copyright [2016]

ObjectivesTo investigate (1) the degree of digital technology adoption among general dental practitioners, and to assess (2) which personal and practice factors are associated with technology use.MethodsA questionnaire was distributed among a stratified sample of 1000 general dental practitioners in the Netherlands, to measure the use of fifteen administrative, communicative, clinical and diagnostic technologies, as well as personal factors and dental practice characteristics.ResultsThe response rate was 31.3%; 65.1% replied to the questionnaire on paper and 34.9% online. Each specific digital technology was used by between 93.2% and 6.8% of the dentists. Administrative technologies were generally used by more dentists than clinical technologies. Dentists had adopted an average number of 6.3±2.3 technologies. 22.5% were low technology users (0 to 4 technologies), 46.2% were intermediate technology users (5 to 7 technologies) and 31.3% were high technology users (8 to12 technologies). High technology users more frequently had a specialization (pConclusionWith few exceptions, all dentists use some or a substantial number of digital technologies. Technology use is associated with various patterns of person-specific factors, and is higher when working in larger dental practices. The findings provide insight into the current state of digital technology adoption in dental practices. Further exploration why some dentists are more reluctant to adopt technologies than others is valuable for the dental profession’s agility in adjusting to technological developments.

High stress stoichiometric silicon nitride resonators, whose quality factors exceed one million, have shown promise for applications in sensing and signal processing. Yet, electrical integration of the insulating silicon nitride resonators has been challenging, as depositing even a thin layer of metal degrades the quality factor significantly. In this work, we show that graphene used as a conductive coating for Si3N4 membranes reduces the quality factor by less than 30 % on average, which is minimal when compared to the effect of conventional metallization layers such as chromium or aluminum. The electrical integration of Si3N4-Graphene (SiNG) heterostructure resonators is demonstrated with electrical readout and electro-static tuning of the frequency by up to 1 % per volt. These studies demonstrate the feasibility of hybrid graphene/nitride mechanical resonators in which the electrical properties of graphene are combined with the superior mechanical performance of silicon nitride., 8 pages, 5 figures, journal

Recent progress in large-area synthesis of monolayer molybdenum disulphide, a new two-dimensional direct-bandgap semiconductor, is paving the way for applications in atomically thin electronics. Little is known, however, about the microstructure of this material. Here we have refined chemical vapour deposition synthesis to grow highly crystalline islands of monolayer molybdenum disulphide up to 120 μm in size with optical and electrical properties comparable or superior to exfoliated samples. Using transmission electron microscopy, we correlate lattice orientation, edge morphology and crystallinity with island shape to demonstrate that triangular islands are single crystals. The crystals merge to form faceted tilt and mirror twin boundaries that are stitched together by lines of 8- and 4-membered rings. Density functional theory reveals localized mid-gap states arising from these 8-4 defects. We find that mirror twin boundaries cause strong photoluminescence quenching whereas tilt boundaries cause strong enhancement. Meanwhile, mirror twin boundaries slightly increase the measured in-plane electrical conductivity, whereas tilt boundaries slightly decrease the conductivity.

Background Digital technologies are proliferating into dental practices. While their technical attributes have often been studied, it remains unclear why some dentists adopt and use these technologies more than others.Aim To explore the incentives for and barriers against accepting and using digital dental technologies.Method Eleven semi-structured qualitative interviews were conducted with experts in dentistry, dental technology and dental education in the Netherlands. Results Dentists' acceptance and use of digital technologies are to varying degrees driven by the perceived advantages over analogue methods, perceived influence on treatment quality, dentists' personal and professional orientation, and social influence from peers and external groups. These effects are complemented by personal and dental-practice characteristics. Conclusions The findings suggest that there are large differences in motivation to adopt and use digital technologies between early adopters, late adopters and non-adopters, which should be examined in greater detail. We recommend that educators, dentists, and representatives of the dental industry who deal with the diffusion of these technologies take account of dentists' widely different attitudes to digitalisation.

Jorge M. Garcia...et al., The growth of single layer graphene nanometer size domains by solid carbon source molecular beam epitaxy on hexagonal boron nitride (h‐BN) flakes is demonstrated. Formation of single‐layer graphene is clearly apparent in Raman spectra which display sharp optical phonon bands. Atomic‐force microscope images and Raman maps reveal that the graphene grown depends on the surface morphology of the h‐BN substrates. The growth is governed by the high mobility of the carbon atoms on the h‐BN surface, in a manner that is consistent with van der Waals epitaxy. The successful growth of graphene layers depends on the substrate temperature, but is independent of the incident flux of carbon atoms., This work is supported by ONR (N000140610138 and Graphene MURI), AFOSR (FA9550‐11‐1‐ 0010), EFRC Center for Re‐Defining Photovoltaic Efficiency through Molecule Scale Control (award DE‐SC0001085), NSF (CHE‐0641523), NYSTAR, CSIC‐PIF (200950I154), Spanish CAM (Q&C Light (S2009ESP‐1503), Numancia 2 (S2009/ENE‐1477)) and Spanish MEC (ENE2009‐14481‐C02‐02, TEC2011‐29120‐C05‐04, MAT2011‐26534).

In chapter 1 of this thesis, an overview of the main applications of NPs was provided and the main properties of NPs were briefly introduced. In addition, some of the key methods that are currently used in the toxicological safety assessment of NPs were presented. The aim of the thesis was introduced which was to investigate the potential of different in vitro methods combined with high-end analytical techniques as a testing strategy to study the toxicokinetic and toxicodynamic properties of silver (Ag) and polystyrene (PS) NPs and set priorities in their further safety testing. The current state of the art of the in vitro methods used in the studies in this thesis and the outline of the thesis were also presented.In chapter 2, the influences of the size and surface chemistry of pristine PSNPs on the protein corona formation and subsequent uptake/association and transport of these NPs through a Caco-2 intestinal cell model were studied. Four negatively charged PSNPs of different sizes (50 and 200 nm) and with different surface chemistries (sulfone or carboxyl groups) were studied. The protein coronas of these PSNPs were analysed by LC-MS/MS which showed subtle differences in the protein composition of especially the two PSNPs with different surface chemistries. In further experiments, the impact of surface chemistry on the cellular uptake/association and transport was characterised using high-content imaging analysis. These experiments demonstrated that the PSNPs with sulfone surface groups were associated with the cells to a significantly higher extent than the PSNPs with carboxyl surface groups. No clear effect of the size of the PSNPs on the cellular uptake/association was noted. Also, the transport of the PSNPs with sulfone surface groups through the monolayer of cells was significantly higher than that of PSNPs with carboxyl surface groups.The general conclusion was that the composition of the protein corona and the PSNPs surface chemistry influence the cellular NPs uptake/association and transport, with the effect of the NP surface chemistry outweighing the impact of NPs size on the cellular uptake/associations and transport. These results might be predictive of the intestinal transport of NPs. Still, further studies are required to identify which corona proteins affect the uptake and transport of NPs.Chapter 3 described an investigation on the impact of the biochemical conditions within the human digestive tract on the intestinal fate of AgNPs with different surface chemistries. On top of that, the transport of these AgNPs across an intestinal in vitro model of Caco-2/HT29-MTX cells was evaluated. An in vitro digestion model was used to simulate the human digestion processes. Two 50 nm negatively charged AgNPs with different surface chemistries were used, lipoic acid (LA) AgNPs and citrate (Cit) AgNPs next to silver nitrate (AgNO3) as a source of ionic silver (Ag+). The co-culture model was exposed to different concentrations of pristine and in vitro digested (IVD) AgNPs or AgNO3 for 24 hr. Using ICP-MS and spICP-MS, the size distribution, dissolution, particle concentration (mass- and number-based) and total silver content of the AgNPs were characterized before and after digestion and in the apical, basolateral and cellular compartments of the Caco-2/HT29-MTX intestinal epithelial model. The surface chemistry of AgNPs had a significant influence on their dissolution and on their biological interactions with the Caco-2/HT29-MTX intestinal model. In general, a significant fraction of the AgNPs dissolved during the digestion up to 86 - 92% and 48 – 70% of the original amount of NPs for the (LA) and (Cit) AgNPs, respectively. Exposure of the monolayer of intestinal cells to increasing concentrations of pristine or IVD AgNPs resulted in a concentration dependent increase of total Ag and AgNPs content in the cellular fractions. The cellular concentrations were significantly lower following exposure to IVD AgNPs compared to the pristine AgNPs. The effect of the surface chemistry on the cellular concentration of Ag was only detected upon exposure to pristine AgNPs, while this difference disappeared upon exposure to IVD AgNPs.Transport of Ag across the intestinal model layer, as either total Ag or AgNPs, was limited (< 0.1%) following exposure to pristine and IVD AgNPs. De novo formation of AgNPs was shown in the exposure suspensions of IVD AgNPs and AgNO3 and in the cellular fractions, upon cellular exposure to pristine and IVD AgNO3. In conclusion, the surface chemistry of AgNPs and the biochemical conditions during in vitro digestion influences the dissolution and also influences the uptake/association within the Caco-2/HT29-MTX monolayer. This highlights the need to take in vitro digestion into account when studying nanoparticle toxicokinetics in an intestinal cellular in vitro model system. The observation that dissolution characteristics of NPs may change upon digestion could be of added value in the safe(r)-by-design NPs development.Chapter 4 presented the outcomes from combining the BeWo b30 placental transport model and the embryonic stem cell test (EST) to investigate the capability of pristine AgNPs of different surface chemistries and aged AgNPs (Ag2S NPs) to cross the placental barrier and induce in vitro developmental toxicity. AgNO3 was used as a source of Ag+. The pristine negatively charged AgNPs used in this study were similar to the ones used in chapter 3 while in addition also positively charged 50 nm branched polyethylenimine (BPEI) AgNPs were included in the study. The size distribution, dissolution, particle concentration (mass- and number-based) and total Ag content of the AgNPs in the apical, basolateral and cellular compartments of the BeWo b30 placental transport model at different time points was characterized using ICP-MS and spICP-MS. The ability of the AgNPs to cross the BeWo b30 cell layer was limited and dependent on the surface chemistry of these AgNPs. The particles detected in the basolateral compartment could result from transport of the original AgNPs and/or from the de novo formed AgNPs in the basolateral compartment from Ag+ that was transported.The in vitro developmental toxicity of the AgNPs was investigated by characterizing their potential to inhibit the differentiation of mouse embryonic stem cells (mESCs) into beating cardiomyocytes using the EST. The observed inhibitory effects of the AgNPs on differentiation of mESCs were most likely the result of cytotoxicity rather than specific effects related to developmental toxicity as the effects on differentiation of the mESCs were only detected at cytotoxic concentrations. Compared to the pristine AgNPs, the aged Ag2S NPs were significantly less cytotoxic, transported less across the BeWo cell layer and did not induce in vitro developmental toxicity. In conclusion, the combination of the BeWo placental transport model with the mESCs differentiation assay appeared to provide a valuable alternative in vitro methodology for prenatal developmental toxicity testing and prioritization for further safety testing of AgNPs, with aged Ag2S NPs appearing to present less of a hazard than pristine AgNPs.In chapter 5, the In-Cell Western (ICW)-γ-H2AX assay was evaluated as an alternative in vitro assay to detect the potential of aged AgNPs and pristine AgNPs to induce phosphorylation of H2AX in HepG2 liver cells. AgNO3 was used as source of Ag+ to test the effects of Ag+ themselves. The γ-H2AX induction detected was higher after 24 hr exposure compared to 4 hr and was accompanied by a significant cytotoxicity in the HepG2 cells. The increased induction of γ-H2AX measured could be due to the cytotoxicity that occurred at the same concentrations which can result in DNA damage resulting in an increased induction of γ-H2AX. This suggests potential false-positive confounders limiting the use of the ICW-γ-H2AX assay, in the form as applied in chapter 5, for evaluation of the genotoxicity of NPs. Additionally, the potential of the AgNPs to induce ROS production, as a potential underlying mechanism of induction of the cytotoxicity and/or DNA-DSBs, was assessed in HepG2 cells. No increase in ROS levels was measured upon exposure of the cells to the AgNPs for 4 or 24 hr and in the absence of cells, while an increase in ROS levels was detected upon AgNO3 exposure of the cells and in the absence of cells.In conclusion, the surface chemistry of AgNPs has a significant influence on their cytotoxic effects and the accompanying induction of γ-H2AX levels in HepG2 cells. The aged Ag2S NPs were biologically less active in inducing both cytotoxicity and γ-H2AX levels, as these effects were absent in the dose range tested. The absence of cellular ROS generation upon exposure to all AgNPs indicates that the observed effects were not ROS-mediated. Additional tests, to rule out apoptotic mediated false-positive signals, need to be combined with the ICW-γ-H2AX assay, to render this interesting assay into a robust screening method for the potential genotoxicity of NPs.Chapter 6 of the present thesis included a general discussion of the results of the previous chapters and highlights on future perspectives for research in the field of in vitro nanotoxicology.Overall, the work presented in this thesis illustrated the role of surface chemistry and the status of the NPs (pristine or aged and/or digested) on the toxicological behaviour of NPs. Besides, the combination of different in vitro models with high-end analytical techniques was shown to; 1) provide in-depth understanding of the biological behaviour of NPs, 2) assure the value of the alternative in vitro models as a testing strategy for potential hazards that could be induced by NPs and 3) assist in setting priorities for in vivo testing and contributing to reduction, refinement and replacement (3Rs) of animal testing required for the safety evaluation of NPs.

Currently, static in vitro cell culture assays are used in early phases of drug development, food research and hazard identification of chemicals. However, these in vitro models lack organ specific functionality, hampering mechanism-based research needed for novel drug development and next generation risk assessment. Recent advances in microchip- and bio-engineering enabled the development of organ-on-a-chip models. The main technical advantages of organ-on-a-chip technology are the ability to spatiotemporally control the microenvironment and the low reagent consumption. On the other hand, micro-engineered organ-on-a-chip models may still lack robustness for reproducible and bio-relevant studies. In addition, also the low flow rates present a major challenge of organ-on-a-chip technology: how to detect drug uptake and compound metabolism in real-time and at a high temporal resolution at the microscale.In this thesis a dynamic intestinal cell culture device is coupled to highly advanced mass spectrometry equipment, aiming for automated and online analysis of translocation of drugs, natural toxins and nanoparticles, while maintaining the bio-integrity of the cell system. Apart from technical robustness challenges, the fundamental chemical incompatibility between complex biological systems used in cell culturing and the clean sample requirements of sensitive advanced analytical instrumentation need to be resolved. Furthermore, the dynamic cell culture model is placed outside of an incubator for the integration with the mass spectrometer. However, physiological relevant temperatures and pH levels still need to be regulated for a reliable biological experiment. As well as the evaluation of the barrier integrity of the intestinal cell layer an aspect largely overlooked by current literature.In Chapter 2 an overview is provided of the current knowledge regarding analytical techniques integrated with organ-on-a-chip systems. In Chapter 3a a dynamic flow-through in vitro model was developed to predict permeability across the intestine using a well-known model drug, verapamil, to benchmark our results against literature. In Chapter 3b the stereoselective permeability of the natural mycotoxin ergotamine was examined using the model established in chapter 3a. The dynamic flow-through in vitro model system was expanded in Chapter 4. A novel bio-integrated online total analysis system for intestinal absorption and metabolism was developed by successfully interfacing a flow-through transwell with electrospray ionization mass spectrometry. The main advantages of this system being the integration of a relevant biological model with fully automated online analysis, which was missing before. Chapter 5 combines a digestion-on-a-chip with the bio-integrated analysis system developed in chapter 4, including the complexity of digestion creating a more complete bio-availability model. In Chapter 6 the integration of the flow-through transwell with inductively coupled plasma mass spectrometry was realized for the evaluation of the translocation of model gold nanoparticles. Lastly, in Chapter 7 a general discussion on the topics described in this thesis is provided and look to the future of organ-on-a-chip technology, with special emphasis on the integration with mass spectrometry analysis.