Application of Impedance-Based Techniques in Hepatology Research

There are a variety of end-point assays and techniques available to monitor hepatic cell cultures and study toxicity within in vitro models. These commonly focus on one aspect of cell metabolism and are often destructive to cells. Impedance-based cellular assays (IBCAs) assess biological functions of cell populations in real-time by measuring electrical impedance, which is the resistance to alternating current caused by the dielectric properties of proliferating of cells. While the uses of IBCA have been widely reported for a number of tissues, specific uses in the study of hepatic cell cultures have not been reported to date. IBCA monitors cellular behaviour throughout experimentation non-invasively without labelling or damage to cell cultures. The data extrapolated from IBCA can be correlated to biological events happening within the cell and therefore may inform drug toxicity studies or other applications within hepatic research. Because tight junctions comprise the blood/biliary barrier in hepatocytes, there are major consequences when these junctions are disrupted, as many pathologies centre around the bile canaliculi and flow of bile out of the liver. The application of IBCA in hepatology provides a unique opportunity to assess cellular polarity and patency of tight junctions, vital to maintaining normal hepatic function. Here, we describe how IBCAs have been applied to measuring the effect of viral infection, drug toxicity/IC50, cholangiopathies, cancer metastasis and monitoring of the gut-liver axis. We also highlight key areas of research where IBCAs could be used in future applications within the field of hepatology

Quantitative Endothelial Cell Monolayer Impedance Sensing and Analysis

The electrical analysis of the biological material has been in existence since the turn of last century. A novel application of this technology to cellular monolayers was implemented by Giaever and Keese 20 years ago with their Electrical Cell-Substrate Impedance Sensing (ECIS) system. The capabilities of a real-time system for endothelial impedance measurement are of immense importance. The endothelium is typically the body’s first contact with stimuli and its reaction to medical conditions of inflammation, disease, and body response are of great significance to understanding the physiology of numerous conditions ranging from heart, lung, and renal disease, to intestinal diseases. It is the purpose of this Master’s thesis to analyze and optimize the ECIS system for making quantitative measurements of endothelial monolayer impedance, and accurately applying the results to a thoroughly reviewed analysis package in order to produce accurate cellular resistance parameters. The optimization of data acquisition (DAQ) is accomplished by systematic noise recognition, examination, and minimization; a task that has previously been unexplored in any studies using the ECIS system. Harmonic, 60 Hz, and Gaussian noise sources were well documented in unfiltered data and successfully minimized in the DAQ. Analog to digital (A/D) noise was found to be the lower limit of reducible noise and was properly documented and considered in analysis. Contamination of the electrode arrays from manufacturing processes and proper electrical connection were also found to be of concern to the proper functioning of the system. Analysis of the optimized acquired data was performed in the LabVIEW programming environment, as it offered a more flexible software package than that provided by the current commercially available ECIS system. The optimized system was applied to a further look into hand arm-vibration syndrome (HAVS) and it was concluded that the acceleration exposure dose, incorrectly calculated from the international standards, did not elicit an acute endothelial inflammation response by our measurements. The cumulative result of this study is that the ECIS system has been optimized and various unresolved sources of error were corrected for a more accurate real-time measurement of the endothelial monolayer barrier function in response to stimuli

EXPLORING THE ROLE AND IMPACT OF MICROSCALE PHENOMENA ON ELECTRODE, MICRODEVICE, AND CELLULAR FUNCTION

Microfluidic technologies enable the development of portable devices to perform multiple high-resolution unit operations with small sample and reagent volumes, low fabrication cost, facile operation, and quick response times. Microfluidic platforms are expected to effectively interpret both wanted and unwanted phenomena; however, a comprehensive evaluation of the unwanted phenomena has not been appropriately investigated in the literature. This work explored an attenuative evaluation of unwanted phenomena, also called here as secondary phenomena, in a unique approach. Upon electric field utilization within microfluidic devices, electrode miniaturization improves device sensitivity. However, electrodes in contact with medium solution can yield byproducts that can change medium properties such as pH as well as bulk ion concentration and eventually target cell viability. While electrode byproducts are sometimes beneficial; but, this is not always the case. Two strategies were employed to protect cells from the electrode byproducts: (i) coating the electrodes with hafnium oxide (HfO2), and (ii) stabilization of the cell membrane using a low concentration of Triton X-100 surfactant. Our results showed that both strategies are a plausible way to selectively isolate cell and reduce the risk of contamination from electrode byproducts. The design of a medium solution is also critical to minimize unwanted cell-medium interaction. Surfactants are frequently added to cell-medium solutions to improve sensitivity and reproducibility without disrupting protein composition of cell membranes or cell viability. In non-electrokinetic systems, surfactants have been shown to reduce interfacial tensions and prevent analyte sticking. However, the impacts of surfactant interactions with cell membranes have not previously been explored in electrokinetic systems. This work indicated the dynamic surfactant interactions with cell membranes which altered the cell membrane integrity. It is important that the effects of the chemical interactions between cells to be fully explored and to be separately attributed to reported cellular responses to accurate catalog properties and engineer reliable microfluidic electrokinetic devices. Finally, a comprehensive level of understanding led us to utilize dielectrophoresis in its full capacity as a tool to monitor the state and progression of virus infection as well as anti-viral activities of regenerative compound. Glycine was utilized as potential antiviral compounds to reduce porcine parvovirus (PPV) infection in porcine kidney (PK-13) cells. Our results demonstrate that the glycine altered the virus-host interactions during virus assembly. Thus, elucidating the mechanisms of these novel antiviral compounds is crucial to their development as potential therapeutic drugs

Using white noise to gate organic transistors for dynamic monitoring of cultured cell layers.

Impedance sensing of biological systems allows for monitoring of cell and tissue properties, including cell-substrate attachment, layer confluence, and the "tightness" of an epithelial tissue. These properties are critical for electrical detection of tissue health and viability in applications such as toxicological screening. Organic transistors based on conducting polymers offer a promising route to efficiently transduce ionic currents to attain high quality impedance spectra, but collection of complete impedance spectra can be time consuming (minutes). By applying uniform white noise at the gate of an organic electrochemical transistor (OECT), and measuring the resulting current noise, we are able to dynamically monitor the impedance and thus integrity of cultured epithelial monolayers. We show that noise sourcing can be used to track rapid monolayer disruption due to compounds which interfere with dynamic polymerization events crucial for maintaining cytoskeletal integrity, and to resolve sub-second alterations to the monolayer integrity

Bacterial sensors and controllers based on organic bioelectronics

Bacterial infections and contaminations are worldwide problems, leading to morbidity and mortality, food waste and economic losses in a variety of industries. The situation is aggravated by the increased occurrence of antibiotic-resistant strains, identified by the WHO as one of the biggest threats to development, food security and public health today. The solution to this problem is complex and requires efforts from several different layers of the society, and different disciplines. The knowledge about microbiology has greatly advanced in the last decades and several powerful methods were introduced. However, in most clinical microbiology laboratories, culture-based techniques are still standard practice, representing a bottleneck in the diagnostic workflow. In this thesis, we prototype novel methods to detect and identify bacteria, aiming to reduce the time and workload for future microbiology research and diagnostics. Furthermore, a new methodology is devised to evaluate antimicrobial surface properties for relevant high-touch surfaces. In Paper I, we investigated whether conducting polymers can be applied for label-free electrochemical detection of bacteria. Employing a poly(3,4-ethylenedioxythiophene): polystyrenesulfonate (PEDOT:PSS)-based two-electrode sensor we demonstrated that potentiometric detection and quantification of Salmonella Enteritidis is possible within 15 min, without any sample pre-treatment. We show that the reduction of PEDOT:PSS electrode occurs by low molecular weight species secreted by Salmonella Enteritidis. To evaluate the genericity of the sensor, several uropathogenic strains were tested and we found that they could all be detected using the sensor. In its current form, the sensor is a prototype, and we aim to improve its sensitivity and introduce specificity. Electroactivity was shown to be a rather common characteristic of bacteria and consequentially, electrochemical methods for detection and characterization of microbes are gaining momentum. We envision that this field will provide novel diagnostic devices but also contribute to discoveries in basic science. Luminescent conjugated oligothiophenes, called optotracers, have previously been applied in microbiology to visualize extracellular matrix components in biofilms of Salmonella and Escherichia coli. In Paper II, we investigated the use of optotracers for detection and visualization of Staphylococcus aureus (S. aureus). We show that the optotracer HS-167 selectively binds to Staphylococci and can be used for fluorometric detection and quantification of S. aureus, as well as for staining and visualization using confocal microscopy. HS-167 displays an on-switch of fluorescence upon binding and it does not affect bacterial growth, which enabled us to develop a high-throughput assay where the fluorescence was plotted against bacterial density, measured as an increase in turbidity. The resulting slope was a quantifiable variable that we employed to compare binding of HS-167 to different species and strains. Diverse approaches collectively pointed to the cell envelope as the target for HS-167 binding. Finally, we showed that binding is highly dependent on the environmental conditions and those can be adjusted to tune the selectivity of HS-167. To improve optotracer design for detection of S. aureus, a better insight into the structure- function relationship is needed. In Paper III, we set out to establish a structured approach to optotracer screening that would enable us to compare optotracer performance. As we compared a library of ten different optotracers, we identified the length to be positively correlated and the total negative charge to be negatively correlated with the ability to detect S. aureus. A balance between the two was necessary to achieve the highest signal while maintaining selectivity. Selected optotracers were added to S. aureus and visualized under the confocal microscope. All localized in the cell envelope of the bacterium, as was previously observed for HS-167 (Paper II). We foresee that further insight into the binding mechanism will enable targeted optotracer design, and together with optimized assay conditions, specific detection of different bacterial species. Copper is known to possess antimicrobial properties, yet studies have reported discrepant results on its efficacy, especially in the clinical settings. Disagreeing results were ascribed to the lack of a standardized approach to evaluate the antimicrobial properties of copper surfaces. In Paper IV, we establish a multifaceted approach to address the effect of human touch, which we simulate by applying artificial sweat, on surface corrosion and antimicrobial properties of copper. We found that artificial sweat accelerates corrosion, leading to changes in surface appearance and wettability. Corrosion did not negatively affect the antimicrobial properties of copper as these surfaces killed bacteria within minutes, regardless of ageing or corrosion product formation. The antimicrobial effect is ascribed in part to copper ions released from the surface and in part to direct surface contact. To further validate the results of this study, other bacterial species need to be tested. Since high touch surfaces are likely to collect a lot of microbes over time, it would be of interest to determine how the bacterial load affects the antimicrobial properties

Design and Characterization of Paper-based Plasma Generators for Sterilization

Nowadays, proper sterilization of surfaces, objects, and even ourselves is a constant need. This work describes the fabrication and thorough characterization of simplistic and disposable plasma-based generators for microbial disinfection, made from metalized paper with aluminum in reference to a previous design developed by Mazzeo et al. These devices rely on the working principle of the dielectric barrier discharge and the generation of plasma was carried out inside a vacuum chamber where different atmospheres were tested. The experimental results demonstrated that additional layers of materials with higher resistance to corrosion and oxidation than aluminum brought no improvements or advantages to reduce the surface degradation by plasmas. These paper-based plasma generators' optical emission spectra indicated that many reactive species desirable in applications such as biological decontamination are produced in the glow discharge. On another note, these generators achieved tolerable biological temperatures (T<40 ºC), representing an excellent approach to the generation of non-thermal plasmas (T<70 ºC). Beyond their characterization, this thesis demonstrated these devices' ability to generate plasma on a reduced scale and a possible optimization method of the decontamination process through plasma confinement. Towards these paper-based generators' stability, they were capable of generating plasma that lasted for more than one hour.Hoje em dia, a esterilização adequada de superfícies, objetos, e mesmo de nós próprios é uma necessidade constante. Este trabalho descreve o fabrico e caracterização minuciosa de geradores de plasma simplistas e descartáveis, para a desinfeção microbiana, feitos de papel metalizado com alumínio em referência a um design previamente desenvolvido por Mazzeo et al. Estes dispositivos baseiam-se no princípio de funcionamento da descarga da barreira dielétrica e a geração de plasma foi realizada dentro de uma câmara de vácuo, onde foram testadas diferentes atmosferas. Os resultados experimentais demonstraram que as camadas adicionais de materiais com maior resistência à corrosão e oxidação do que o alumínio não trouxeram melhorias ou vantagens na redução da degradação da superfície pelo plasma. Os espectros de emissão ótica destes geradores indicaram que muitas das espécies reativas desejáveis em aplicações tais como a descontaminação biológica são produzidas na descarga luminescente. Por outro lado, estes geradores alcançaram temperaturas biológicas toleráveis (T<40 ºC), representando uma excelente abordagem à geração de plasmas não térmicos (T<70 ºC). Para além da caracterização destes geradores, esta tese demonstrou a capacidade destes dispositivos de gerar plasma a uma escala reduzida e um possível método de otimização do processo de descontaminação através do confinamento do plasma. Relativamente à estabilidade destes geradores de plasma em papel, estes foram capazes de gerar plasma com uma durabilidade superior a uma hora

Selected Papers from the 1st International Electronic Conference on Biosensors (IECB 2020)

The scope of this Special Issue is to collect some of the contributions to the First International Electronic Conference on Biosensors, which was held to bring together well-known experts currently working in biosensor technologies from around the globe, and to provide an online forum for presenting and discussing new results. The world of biosensors is definitively a versatile and universally applicable one, as demonstrated by the wide range of topics which were addressed at the Conference, such as: bioengineered and biomimetic receptors; microfluidics for biosensing; biosensors for emergency situations; nanotechnologies and nanomaterials for biosensors; intra- and extracellular biosensing; and advanced applications in clinical, environmental, food safety, and cultural heritage fields

Nature’s Optics and Our Understanding of Light

Optical phenomena visible to everyone abundantly illustrate important ideas in science and mathematics. The phenomena considered include rainbows, sparkling reflections on water, green flashes, earthlight on the moon, glories, daylight, crystals, and the squint moon. The concepts include refraction, wave interference, numerical experiments, asymptotics, Regge poles, polarisation singularities, conical intersections, and visual illusions

The Use of Skeletal Muscle to Amplify Action Potentials in Transected Peripheral Nerves

Upper limb amputees suffer with problems associated with control and attachment of prostheses. Skin-surface electrodes placed over the stump, which detect myoelectric signals, are traditionally used to control hand movements. However, this method is unintuitive, the electrodes lift-off, and signal selectivity can be an issue. One solution to these limitations is to implant electrodes directly on muscles. Another approach is to implant electrodes directly into the nerves that innervate the muscles. A significant challenge with both solutions is the reliable transmission of biosignals across the skin barrier. In this thesis, I investigated the use of implantable muscle electrodes in an ovine model using myoelectrodes in combination with a bone-anchor, acting as a conduit for signal transmission. High-quality readings were obtained which were significantly better than skin-surface electrode readings. I further investigated the effect of electrode configurations to achieve the best signal quality. For direct recording from nerves, I tested the effect of adsorbed endoneural basement membrane proteins on nerve regeneration in vivo using microchannel neural interfaces implanted in rat sciatic nerves. Muscle and nerve signal recordings were obtained and improvements in sciatic nerve function were observed. Direct skeletal fixation of a prosthesis to the amputation stump using a bone-anchor has been proposed as a solution to skin problems associated with traditional socket-type prostheses. However, there remains a concern about the risk of infection between the implant and skin. Achieving a durable seal at this interface is therefore crucial, which formed the final part of the thesis. Bone-anchors were optimised for surface pore size and coatings to facilitate binding of human dermal fibroblasts to optimise skin-implant seal in an ovine model. Implants silanised with Arginine-Glycine-Aspartic Acid experienced significantly increased dermal tissue infiltration. This approach may therefore improve the soft tissue seal, and thus success of bone-anchored implants. By addressing both the way prostheses are attached to the amputation stump, by way of direct skeletal fixation, as well as providing high fidelity biosignals for high-level intuitive prosthetic control, I aim to further the field of limb loss rehabilitation

A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond

According to the World Health Organization (WHO), almost 2 billion people each year are infected worldwide with flu-like pathogens including influenza. This is a contagious disease caused by viruses belonging to the family Orthomyxoviridae. Employee absenteeism caused by flu infection costs hundreds of millions of dollars every year. To successfully treat influenza virus infections, detection of the virus during the initial development phase of the infection is critical, when tens to hundreds of virus-associated molecules are present in the patient’s pharynx. In this study, we describe a novel universal diamond biosensor, which enables the specific detection of the virus at ultralow concentrations, even before any clinical symptoms arise. A diamond electrode is surface-functionalized with polyclonal anti-M1 antibodies, which then serve to identify the universal biomarker for the influenza virus, M1 protein. The absorption of the M1 protein onto anti-M1 sites of the electrode change its electrochemical impedance spectra. We achieved a limit of detection of 1 fg/ml in saliva buffer for the M1 biomarker, which corresponds to 5–10 viruses per sample in 5 minutes. Furthermore, the universality of the assay was confirmed by analyzing different strains of influenza A virus