A Label Free CMOS-Based Smart Petri Dish for Cellular Analysis

RÉSUMÉ Le dépistage de culture cellulaire à haut débit est le principal défi pour une variété d’applications des sciences de la vie, y compris la découverte de nouveaux médicaments et le suivi de la cytotoxicité. L’analyse classique de culture cellulaire est généralement réalisée à l’aide de techniques microscopiques non-intégrées avec le système de culture cellulaire. Celles-ci sont laborieuses spécialement dans le cas des données recueillies en temps réel ou à des fins de surveillance continue. Récemment, les micro-réseaux cellulaires in-vitro ont prouvé de nombreux avantages dans le domaine de surveillance des cellules en réduisant les coûts, le temps et la nécessité d’études sur des modèles animaux. Les microtechniques, y compris la microélectronique et la microfluidique,ont été récemment utilisé dans la biotechnologie pour la miniaturisation des systèmes biologiques et analytiques. Malgré les nombreux efforts consacrés au développement de dispositifs microfluidiques basés sur les techniques de microscopie optique, le développement de capteurs intégrés couplés à des micropuits pour le suivi des paramètres cellulaires tel que la viabilité, le taux de croissance et cytotoxicité a été limité. Parmi les différentes méthodes de détection disponibles, les techniques capacitives offrent une plateforme de faible complexité. Celles-ci ont été considérablement utilisées afin d’étudier l’interaction cellule-surface. Ce type d’interaction est le plus considéré dans la majorité des études biologiques. L’objectif de cette thèse est de trouver des nouvelles approches pour le suivi de la croissance cellulaire et la surveillance de la cytotoxicité à l’aide d’un réseau de capteurs capacitifs entièrement intégré. Une plateforme hybride combinant un circuit microélectronique et une structure microfluidique est proposée pour des applications de détection de cellules et de découverte de nouveaux médicaments. Les techniques biologiques et chimiques nécessaires au fonctionnement de cette plateforme sont aussi proposées. La technologie submicroniques Standard complementary metal-oxide-Semiconductor (CMOS) (TSMC 0.35 μm) est utilisée pour la conception du circuit microélectronique de cette plateforme. En outre, les électrodes sont fabriquées selon le processus CMOS standard sans la nécessité d’étapes de post-traitement supplémentaires. Ceci rend la plateforme proposée unique par rapport aux plateformes de dépistage de culture cellulaire à haut débit existantes. Plusieurs défis ont été identifiés durant le développement de cette plateforme comme la sensibilité, la bio-compatibilité et la stabilité et les solutions correspondantes sont fournies.----------ABSTRACT High throughput cell culture screening is a key challenge for a variety of life science applications, including drug discovery and cytotoxicity monitoring. Conventional cell culture analysis is widely performed using microscopic techniques that are not integrated into the target cell culture system. Additionally, these techniques are too laborious in particular to be used for real-time and continuous monitoring purposes. Recently, it has been proved that invitro cell microarrays offer great advantages for cell monitoring applications by reducing cost, time, and the need for animal model studies. Microtechnologies, including microelectronics and microfluidics, have been recently used in biotechnology for miniaturization of biological and analytical systems. Despite many efforts in developing microfluidic devices using optical microscopy techniques, less attention have been paid on developing fully integrated sensors for monitoring cell parameters such as viability, growth rate, and cytotoxicity. Among various available sensing methods, capacitive techniques offer low complexity platforms. This technique has significantly attracted attentions for the study of cell-surface interaction which is widely considered in biological studies. This thesis focuses on new approaches for cell growth and cytotoxicity monitoring using a fully integrated capacitive sensor array. A hybrid platform combining microelectronic circuitry and microfluidic structure is proposed along with other required biological and chemical techniques for single cell detection and drug discovery applications. Standard submicron complementary metal–oxide–semiconductor (CMOS) technology (TSMC 0.35 μm) is used to develop the microelectronic part of this platform. Also, the sensing electrodes are fabricated in standard CMOS process without the need for any additional post processing step, which makes the proposed platform unique compared to other state of the art high throughput cell assays. Several challenges in implementing this platform such as sensitivity, bio-compatibility, and stability are discussed and corresponding solutions are provided. Specifically, a new surface functionalization method based on polyelectrolyte multilayers deposition is proposed to enhance cell-electrode adherence and to increase sensing electrodes’ life time. In addition, a novel technique for microwell fabrication and its integration with the CMOS chip is proposed to allow parallel screening of cells. With the potential to perform inexpensive, fast, and real-time cell analyses, the proposed platform opens up the possibility to transform from passive traditional cell assays to a smart on-line monitoring system

Electrochemical enzyme-based biosensor array for monitoring of organic acids and ethanol in biogas processes

In light of steadily increasing energy demand and irreversible exhaustion of fossil fuels, further expansion of renewable energy sources is continually gaining importance. Utilization of biomass, as a widely available energy carrier, is capable of providing great contribution to sustainable energy supply. The efficient production of biogas, however, calls for an improved biomass supply chain. Economic operation of biogas plants depends in particular on reliable process monitoring. Often, process disturbances are accompanied by fluctuations in the concentration profile of some intermediates produced during the anaerobic fermentation process. Nowadays, the focus has mainly been set on volatile fatty acids (such as acetate and propionate) as an indicator for imbalanced process conditions and only little account has been taken to the relevance of other organic acids and alcohols, like lactate, formate and ethanol. In this work, an electrochemical enzyme-based biosensor array for simultaneous determination of D-lactate, L-lactate, formate and ethanol is developed. The amperometric sensing principle is based on two enzymes in each case: an analyte-specific NAD+-dependent dehydrogenase combined with a diaphorase from Clostridium kluyveri. The latter converts its substrate Fe(CN)63- to Fe(CN)64-, which generates a concentration-dependent current by oxidation at an polarized electrode. Enzymes were immobilized by chemical cross-linking with glutaraldehyde on platinum thin-film electrodes. The optimization of the biosensor performance has been investigated in regard to enzyme loading, glutaraldehyde concentration, cofactor concentration (NAD+ and Fe(CN)63-), pH value and temperature. The potential for repeated and long-term application has been proven by evaluation of operational and storage stability. Typically, enzyme-based biosensors are characterized by a high specificity due to the remarkable properties of enzymes as biological recognition element. Measurements in real samples, however, are prone to interfering effects by other electroactive species in the sample solution. The specificity of the biosensing system is determined in response to various interfering compounds and results reveal no cross-talk effects during simultaneous measurement of the four different analytes of interest. Successful practical performance for rapid and on-site analysis, has been demonstrated by quantification of D-lactate, L-lactate, formate and ethanol in various feedstocks (maize- and sugar cane silage) and spiked fermentation samples from three industrial biogas plants. Good correlation is obtained for results determined by the biosensor array in comparison to conventional commercial analytical methods applied (photometry and gas chromatography). In contrast to these techniques, the biosensor array offers the advantages of facile on-site application with a portable measurement set-up, rapid analysis time by simultaneous operation and application in untreated samples. The measuring system has also been applied for long-term monitoring of a lab-scale biogas reactor (0.01 m3) for a period of two months. Regular analysis of alcohol- and organic acid levels provides a beneficial supplementation to standard monitoring parameters, like biogas production, methane yield, pH and temperature. This additional information can help to identify changes in the microbial methane formation and potentially indicate upcoming imbalances at an early stage. For improved practical implementation of the developed biosensor array, the required cofactors have been co-immobilized on the sensor surface of screen-printed carbon electrodes. Modification with graphene oxide enables the establishment of a reagent-free biosensing system. Such biosensors can be manufactured economically by thick-film technology and used as disposable test strips for simplified on-site monitoring of several key intermediates in the biogas fermentation medium

Correlating the Effect of Dynamic Variability in the Sensor Environment on Sensor Design

This dissertation studies the effect of biofluid dynamics on the electrochemical response of a wearable sensor for monitoring of chronic wounds. The research investigates various dynamic in vivo parameters and correlates them with experimentally measured behavior with wound monitoring as a use case. Wearable electrochemical biosensors suffer from several unaddressed challenges, like stability and sensitivity, that need to be resolved for obtaining accurate data. One of the major challenges in the use of these sensors is continuous variation in biofluid composition. Wound healing is a dynamic process with wound composition changing continuously. This dissertation investigates the effects of several in vivo biochemical and environmental parameters on the sensor response to establish actionable correlations. Real-time assessment of wound healing was carried out through longitudinal monitoring of uric acid and other wound fluid characteristics. A textile sensor was designed using a simple fabrication approach combining conductive inks with a polymeric substrate, for conformal contact with the wound bed. A −1 cm−2, establishing the applicability of the sensor for measurements in the physiologically relevant range. The sensor was also found to be stable for a period of 3 days when subjected to physiological and elevated temperatures (37oC and 40oC) confirming its relevance for long-term monitoring. A direct correlation between sensor response and the dynamic parameters was seen, with the results showing a ~20% deviation from the accurate UA reading. The results confirmed that as a consequence of these parameters temporally changing in the wound environment, the sensor response will be altered. The work develops mathematical models correlating this effect on sensor response to allow for real-time sensor calibration. The clinical validation studies established the feasibility of UA measurement by the developed electrochemical sensor and derive correlations between the wound chronicity and UA levels. The protocols developed in this work for the design, fabrication, and calibration of the sensor to correct for the dynamic in vivo behavior can be extended to any wearable sensor for improved accuracy

Investigation of light-addressable potentiometric sensors for electrochemical imaging based on different semiconductor substrates

PhDLight-addressable potentiometric sensors (LAPS) and scanning photo-induced impedance microscopy (SPIM) have been extensively applied as chemical sensors and biosensors. This thesis focuses on the investigation of LAPS and SPIM for electrochemical imaging based on two different semiconductor substrates, silicon on sapphire (SOS) and indium tin oxide (ITO) coated glass. Firstly, SOS substrates were modified with 1,8-nonadiyne self-assembled organic monolayers (SAMs), which served as the insulator. The resultant alkyne terminals provided a platform for the further functionalization of the sensor substrate with various chemicals and biomolecules by Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) ‘click’ reactions. The CuAAC reaction combined with microcontact printing (μCP) was successfully used to create chemical patterns on alkyne-terminated SOS substrates. The patterned monolayers were found to be contaminated with the copper catalyst used in the click reaction as visualized by LAPS and SPIM. Different strategies for avoiding copper contamination were tested. Only cleaning of the silicon surfaces with an ethylenediaminetetraacetic acid tetrasodium salt (EDTA) solution containing trifluoroacetic acid after the ‘click’ modification proved to be an effective method as confirmed by LAPS and SPIM results, which allowed, for the first time, the impedance of an organic monolayer to be imaged. Furthermore, the 1,8-nonadiyne modified SOS substrate was functionalized and patterned with an RGD containing peptide, which was used to improve the biocompatibility of the substrate and cell adhesion. By seeding cells on the peptide patterned sensor substrate, cell patterning was achieved. Single cell imaging using LAPS and SPIM was attempted on the RGD containing peptide modified SOS substrate Finally, an ITO coated glass substrate was used as a LAPS substrate for the first time. The photocurrent response, the pH response, LAPS and SPIM imaging and its lateral resolution using ITO coated glass without any modification were investigated. Importantly, single cell images were obtained with this ITO-based LAPS systemChina Scholarship Council and Queen Mary University of Londo

Field-Effect Sensors

This Special Issue focuses on fundamental and applied research on different types of field-effect chemical sensors and biosensors. The topics include device concepts for field-effect sensors, their modeling, and theory as well as fabrication strategies. Field-effect sensors for biomedical analysis, food control, environmental monitoring, and the recording of neuronal and cell-based signals are discussed, among other factors

Functionalized graphene sensors for real time monitoring fermentation processes:electrochemical and chemiresistive sensors

We developed a reference-less, chemiresistive, solid-state pH sensor to determine the acidification of the fermentation liquid in real time during the growth of Lactococcus lactis. One of the crucial findings of this work was that the ERGO-PA could not be used as such. It appeared that it was necessary to protect the sensor area with a Nafion coating to measure the pH in the fermentation broth. Most likely, the change in the concentration of redox-active components in the fermentation broth influences the conductivity of the ERGO-PA. Nafion formed a cation-selective membrane on top of the ERGO-PA allowing protons to diffuse to the selective layer of the sensor but not the redox-active components in the fermentation medium. We also reported a new approach to measure the dissolved oxygen concentration (DO) in a fermentation broth. The functionality of the sensor to measure DO was demonstrated during the growth of the obligate aerobic actinomycete Amycalotopsis methanolica in miniaturized 3D-printed bioreactors. For this oxygen-sensing application, the required modifications were obtained by doping hydrothermally reduced graphene oxide with nitrogen and boron atoms (N,B-HRGO). Further, these chemiresistive sensors are housed in the 3D printed bioreactor lid and used to measure pH, DO, and biomass in 3 ml fermentation broth. Additionally, the pH-sensor was equipped with a small heating element and a temperature sensor and that could be used for temperature control of the fermentation liquid. The setup was demonstrated to measure the pH, DO, temperature and biomass concentration in four parallel bioreactors

New biosensors for metabolic imaging

Tissue engineering is a multi-disciplinary field that involves three-dimensional cell and tissue models, live cell microscopy and related imaging modalities, along with fluorescence and phosphorescence-based biosensors. These technologies can work together in developing biologically relevant 3D tissue models for the modelling of complex physiological and diseased states. One of the main challenges facing such models is the lack of non-invasive strategies for quantitative real time monitoring of cellular and tissue physiology, metabolism and viability, that are compatible with live cell microscopy. This thesis presents the design and development of new biosensor, scaffold and nanoparticle materials, with the aim of facilitating quantitative metabolic imaging in cell and tissue culture, via fluorescence lifetime microscopy and phosphorescence lifetime microscopy. Thus, we have developed protein-based biosensor probes, sensitive to pH and calcium in intensity and fluorescence lifetime modalities for the labelling of cellulose scaffold materials, producing a hybrid scaffold material for tissue engineering applications. This was done by genetically engineering of recombinant proteins expressing the cellulose-binding domain (CBD) CenA protein, derived from the fungus C. fimi, fused to pH-sensitive enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), forming CBD-ECFP and CBD-EYFP biosensors, respectively. A third biosensor was also developed with CBD and the genetically encoded calcium indicator known as circularly permutated EGFP (cpEGFP)/M13/Calmodulin (CaM) fusion protein (GCaMP2) forming CBD-GCaMP2. For all three CBD constructs we observed responses in fluorescence intensity to changes in calcium for GCaMP2 and pH for both CBD-ECFP and CBD-EYFP, achieving efficient and stable labelling of various cellulose scaffolds including nanofibrillar, GrowDex, bacterial cellulose and decellularised plant materials. CBD-ECFP labelled GrowDex produced a biosensor scaffold material capable of supporting the growth of 3D cultured human colon cancer cells HCT116, with the ability to measure real-time changes in extracellular pH. The developed labelling strategy allows for the design of biosensor scaffold materials with potential multi-parametric fluorescence lifetime microscopy modalities, which can be used to achieve the controlled production of 3D tissue models with measurable pH and metabolic gradients. Intracellular metabolic imaging is currently dominated by synthetic nanoparticle constructs that suffer from suboptimal intracellular staining, along with high toxicity and immunogenicity. Here we developed several self-assembling protein nanoparticle constructs based on viral like particle, elastin like polypeptide-cowpea chlorotic mottle virus capsid protein (ELP-CP) and protein nanocage ferritin. Such constructs hold promise due to their biological nature making them more biocompatible and biodegradable, thereby reducing toxic and immunogenic effects. Such self-assembling protein nanoparticles are also amenable to multiple strategies of functionalisation such as metallochelate coupling, genetic engineering, chemical modification, and encapsulation. We evaluated metallochelate coupling to design intracellular O2-sensitive biosensors, where oligohistidine-tagged recombinant proteins are bound to nitrilotriacetate (NTA) or iminodiacetic acid (IDA) groups on dyes and small molecules. The NTA or IDA groups form a complex with transition metal ions such as: Zn2+, Ni2+, Co2+, or Cu2+. This complex then co-ordinates to histidine amino acids on the recombinant protein. We successfully produced ratiometric phosphorescent probes from enhanced green fluorescent protein (EGFP), enhanced monomeric blue fluorescent protein 2 (mTagBFP2) and Discosoma red fluorescent protein (DsRed express) coupled to tetracarboxylic platinum (II)-coproporphyrin I (PtCP) PtCP-NTA. Such complexes can be used for ratiometric-based measurements of O2, where fluorescent proteins (FPs) can be used as O2-insensitive references. Most notably we demonstrated the first example of a phosphorescent O2-senstive viral like particle (VLP) structure, ELPCP-H6-PtCP and in comparing to commercial O2-sensitive probe MitoXpress, we observed higher phosphorescence brightness, similar lifetime responses and increased sensitivity in response to O2. The potential to couple a range of FPs or self-assembling protein nanoparticles to O2 sensitive phosphorescent dyes demonstrates that metallochelate coupling is a highly attractive strategy in the design of new intracellular O2 sensors. Using genetic engineering and encapsulation strategies we successful produced both pH and O2- sensitive ferritin nanoparticles. Genetic engineering enabled the expression of multiple cell targeting and penetrating peptides, such as bactenecin 7 and α-enolase, along with fluorescent proteins EGFP or ECFP, without affecting spectral properties of the fluorescent proteins or ferritin self-assembly. Genetic engineered ECFP-FTN construct demonstrated pH sensitivity in fluorescence intensity and lifetime across a physiological range of pH, potentially allowing for applications in fluorescence lifetime microscopy-based measurements of intracellular pH. Through the strategy of pH dependent disassembly and reassembly encapsulation of phosphorescent O2 sensitive probe Pt-Glc, we successfully produced O2-sensitive horse ferritin-based (hoFTN) nanoparticles. The resulting hoFTNPt-Glc displayed a higher phosphorescence intensity signal than free Pt-Glc, possibly due to the concentrated number of Pt-Glc molecules in close proximity within the ferritin structure, and demonstrated responses to oxygenation, increasing phosphorescence intensity when deoxygenated. However, in characterisation of hoFTN-Pt-Glc with MEF cells we observed poor intracellular staining confined to endosomes, similar to free Pt-Glc. These results showed that encapsulation here does not improve intracellular staining or phosphorescence lifetime responses. Despite poor characterisation of ferritin constructs in HCT116 and MEF cell lines, the strategies evaluated here show promise and demonstrate an interchangeable approach to functionalising self-assembling protein nanoparticles and fluorescent proteins for applications in fluorescence lifetime microscopy and phosphorescence lifetime microscopy-based quantitative and ratiometric live cell imaging

Biosensor system with an integrated CMOS microelectrode array for high spatio-temporal electrochemical imaging, A

2019 Fall.Includes bibliographical references.The ability to view biological events in real time has contributed significantly to research in life sciences. While optical microscopy is important to observe anatomical and morphological changes, it is equally important to capture real-time two-dimensional (2D) chemical activities that drive the bio-sample behaviors. The existing chemical sensing methods (i.e. optical photoluminescence, magnetic resonance, and scanning electrochemical), are well-established and optimized for existing ex vivo or in vitro analyses. However, such methods also present various limitations in resolution, real-time performance, and costs. Electrochemical method has been advantageous to life sciences by supporting studies and discoveries in neurotransmitter signaling and metabolic activities in biological samples. In the meantime, the integration of Microelectrode Array (MEA) and Complementary-Metal-Oxide-Semiconductor (CMOS) technology to the electrochemical method provides biosensing capabilities with high spatial and temporal resolutions. This work discusses three related subtopics in this specific order: improvements to an electrochemical imaging system with 8,192 sensing points for neurotransmitter sensing; comprehensive design processes of an electrochemical imaging system with 16,064 sensing points based on the previous system; and the application of the system for imaging oxygen concentration gradients in metabolizing bovine oocytes. The first attempt of high spatial electrochemical imaging was based on an integrated CMOS microchip with 8,192 configurable Pt surface electrodes, on-chip potentiostat, on-chip control logic, and a microfluidic device designed to support ex vivo tissue experimentation. Using norepinephrine as a target analyte for proof of concept, the system is capable of differentiating concentrations of norepinephrine as low as 8µM and up to 1,024 µM with a linear response and a spatial resolution of 25.5×30.4μm. Electrochemical imaging was performed using murine adrenal tissue as a biological model and successfully showed caffeine-stimulated release of catecholamines from live slices of adrenal tissue with desired spatial and temporal resolutions. This system demonstrates the capability of an electrochemical imaging system capable of capturing changes in chemical gradients in live tissue slices. An enhanced system was designed and implemented in a CMOS microchip based on the previous generation. The enhanced CMOS microchip has an expanded sensing area of 3.6×3.6mm containing 16,064 Pt electrodes and the associated 16,064 integrated read channels. The novel three-electrode electrochemical sensor system designed at 27.5×27.5µm pitch enables spatially dense cellular level chemical gradient imaging. The noise level of the on-chip read channels allow amperometric linear detection of neurotransmitter (norepinephrine) concentrations from 4µM to 512µM with 4.7pA/µM sensitivity (R=0.98). Electrochemical response to dissolved oxygen concentration or oxygen partial pressure (pO2) was also characterized with deoxygenated deionized water containing 10µM to 165 µM pO2 with 8.21pA/µM sensitivity (R=0.89). The enhanced biosensor system also demonstrates selectivity to different target analytes using cyclic voltammetry to simultaneously detect NE and uric acid. In addition, a custom-designed indium tin oxide and Au glass electrode is integrated into the microfluidic support system to enable pH measurement, ensuring viability of bio-samples in ex vivo experiments. Electrochemical images confirm the spatiotemporal performance at four frames per second while maintaining the sensitivity to target analytes. The overall system is controlled and continuously monitored by a custom-designed user interface, which is optimized for real-time high spatiotemporal resolution chemical bioimaging. It is well known that physiological events related to oxygen concentration gradients provide valuable information to determine the state of metabolizing biological cells. Utilizing the CMOS microchip with 16,064 Pt MEA and an improved three-electrode system configuration, the system is capable of imaging low oxygen concentration with limit of detection of 18.3µM, 0.58mg/L, or 13.8mmHg. A modified microfluidic support system allows convenient bio-sample handling and delivery to the MEA surface for sensing. In vitro oxygen imaging experiments were performed using bovine cumulus-oocytes-complexes cells with custom software algorithms to analyze its flux density and oxygen consumption rate. The imaging results are processed and presented as 2D heatmaps, representing the dissolved oxygen concentration in the immediate proximity of the cell. The 2D images and analysis of oxygen consumption provide a unique insight into the spatial and temporal dynamics of cell metabolism