Biomimetic Yeast Cell Typing—Application of QCMs

Artificial antibodies represent a key factor in the generation of sensing systems for the selective detection of bioanalytes of variable sizes. With biomimetic surfaces, the important model organism Saccharomyces cerevisiae and several of its growth stages may be detected. Quartz crystal microbalances (QCM) with 10 MHz fundamental frequency and coated with polymers imprinted with synchronized yeast cells are presented, which are able to detect duplex cells with high selectivity. Furthermore, a multichannel quartz crystal microbalance (MQCM) was designed and optimized for the measurement in liquids. This one-chip system based on four-electrode geometry allows the simultaneous detection of four analytes and, thus, provides a monitoring system for biotechnology and process control. For further standardization of the method, synthetic stamps containing plastic yeast cells in different growth stages were produced and utilized for imprinting. Mass-sensitive measurements with such MIPs resulted in the same sensor characteristics as obtained for those imprinted with native yeast cells

Molecularly Imprinted Polymers for Cell Recognition

Since their conception 50 years ago, molecularly imprinted polymers (MIPs) have seen extensive development both in terms of synthetic routes and applications. Cells are perhaps the most challenging target for molecular imprinting. Although early work was based almost entirely around microprinting methods, recent developments have shifted towards epitope imprinting to generate MIP nanoparticles (NPs). Simultaneously, the development of techniques such as solid phase MIP synthesis has solved many historic issues of MIP production. This review briefly describes various approaches used in cell imprinting with a focus on applications of the created materials in imaging, drug delivery, diagnostics, and tissue engineering

Modellierung von Bioanalyten durch Polymere und deren Verwendung zur Strukturierung von Oberflächen

Die Schnellanalytik von Bioanalyten kann mit massensensitiven Sensoren durchgeführt werden. Hierzu verwendet man strukturierte Oberflächen, die selektiv z.B. Viren und Zellen aus der Lösung anreichern können. Vorteilhaft ist bei dieser Strategie, dass kein Labelling notwendig ist, da die massensensitive Detektion ein generelles Verfahren darstellt. Entscheidend ist hierfür die Optimierung der Selektivität, die über die Geometrie der Biopartikel und die Wechselwirkung zwischen Schicht und Analyt erreicht werden kann. Engpass bei einem derartigen Vorgehen ist die Variabilität und Degradation von Bioanalyten. Zellen unterliegen einem Wachstumsprozess und infolge der Nährlösung laufen unterschiedliche Veränderungen ab. Hervorzuheben sind insbesondere der Mitosestatus, Knospungen und die Bildung von Dimeren wie dem S-Zustand. Zur Standardisierung sind deshalb Plastik-Zellen von Interesse, die in eindeutiger Weise einen definierten Typus charakterisieren. Zu deren Herstellung werden Polymere mit den jeweiligen Zellen über eine Stempelmethode strukturiert und die Hohlräume mit einem anderen Polymer ausgegossen. Mit diesem synthetischen Stempel können dann standardisierte Prägungen durchgeführt werden. Das Problem bei dieser Methode ist die Trennung von Stempel und Oberfläche, was durch Variation der Polymere erreicht werden soll. Insbesondere ist hierbei das Ausmaß der Wasserstoffbrückenbindungen entscheidend

QCM sensor arrays for monitoring volatile plant emanations via molecularly imprinted polymers

Zur Überwachung flüchtiger organischer Verbindungen (VOC volatile organic component) wurde ein Sensorarray bestehend aus mehrkanaligen Schwingquarzen (MQCM multichannel quartz crystal microbalance) entwickelt. Als selektive Schicht für den jeweiligen Analyten kamen molekular geprägte Polymerbeschichtungen zum Einsatz. Diese Sensor Arrays wurden vorerst zur kontinuierlichen online Überwachung und selektiven Quantifizierung von Terpenen eingesetzt, die von Arten der Familie Lamiaceae, wie beispielsweise Pfefferminze (Mentha x piperinta) und Basilikum (Ocimum Basilicum) freigesetzt werden. Dabei erzielten die Sensoren bei der Bestimmung des Frischegrades bemerkenswerte Reproduzierbarkeit der Emanationsmuster. Diese waren vergleichbar zu GC-MS Messungen, mit einem Detektionslimit unter 70 ppm. Zusätzlich können diese Muster mit Erkennungsmethoden wie zum Beispiel der Hauptkomponentenanalyse (PCA principal component analyses), der PLS (partial least squares) und mittels künstlichen neuronalen Netzwerken (ANN artifical neuronal networks) untersucht werden. Des Weiteren wurde eine elektronische Nase mit einer molekular geprägten, biomimetischen Polymerschicht (MIP molecular imprinted polymer) entwickelt, die das Terpenemissionsmuster von frischen und getrockneten Kräutern charakterisiert. Hierzu wurden Rosmarin (Rosmarinus Officinalis L.), Basilikum (Ocimum Basilicum) und Salbei (Salvia Officinalis) eingesetzt. Die dazu notwendigen Optimierungsparamater der elektronischen Nase sind: Schichtdicken, Sensitivität der Analyte <20 ppm, Selektivität bei einer Konzentration von 50 ppm der Terpene und Reproduzierbarkeit. Die reversiblen Sensorantworten sind in einem Konzentrationsbereich von <20 ppm bis 200 ppm linear. Die Isomere α- und β-Pinen sind signifikant unterscheidbar. Die Unterscheidbarkeit zwischen frischen und getrockneten Kräutern konnte durch die entsprechenden Messeffekte (20-1200 Hz) der elektronischen Nasen realisiert werden. Die erhaltenen Daten wurden zur Mustererkennung mittels PCA und ANN analysiert und durch Ergebnisse der GC-MS Messungen, welche einen ähnlichen Trend darstellen, validiert. Die Haltbarkeitsdauer der Kräuter konnte durch die Emanation der flüchtigen organischen Bestandteile über einen Zeitraum von mehreren Tagen bestimmt werden. Die Detektionslimits sind besser als 20 ppm und erlauben die Überwachung der Lagerung über mehrere Tage. Zusätzlich wurde eine MIP Screening Methode zur chemischen Bestimmung von Ethlyacetat entwickelt. Dazu wurden sechs MIPs mit unterschiedlichen Monomer¬zusammensetzungen aus VP, PS und DVB hergestellt und getestet. Als das am besten geeignete Sensormaterial für Ethylacetat erwies sich das Polymer mit einer Monomer¬zusam¬mensetzung von VP:PS:DVB 1:2:7 etabliert. Damit konnten Sensitivitäten und Selektivitäten über einen weiten Konzentrationsbereich von 25-3000 ppm für Ethylacetat erreicht werden. Die Querselektivität dieses MIP zwischen 250 und 750 ppm zu 1-Propanol erwies sich als sehr gering (≤ 1 Hz). Schlussendlich wurde ein Sensor Array mit vier Elektroden pro Substrat konstruiert. Dessen Herstellung wurde durch die Optimierung der Elektrodengröße, deren Geometrie und dem Kalibrieren der Heizwendel bestimmt. Diese neu entwickelte Strategie wurde zur massensensitiven real-time Bestimmung und Unterscheidung von Terpenen, welche von Thymian freigesetzt werden, eingesetzt. Die Muster der freigesetzten Terpene, welche mittels vier-Elektroden QCM Arrays erhalten wurden, sind mit den GC-MS Daten vergleichbar. Somit können derartige QCM Sensorarrays in der Praxis zur sensitiven und selektiven Bestimmung einer Vielzahl von biologischen Analyten im mikro- und makro-Bereich, wie beispielsweise die DNA Bestimmung, die Überwachung von VOCs von Pflanzen, bei der Kompostierung oder bei Abbauprozessen, die Qualitätskontrolle, die Haltbarkeit und Frische von Lebensmittel und in zahlreichen Industriebereichen, in unterschiedlichen Phasen zum Einsatz kommen.Arrays of chemical sensors derived from 10 MHz piezoelectric multichannel quartz crystal microbalance (MQCM) have been developed for selective monitoring of volatile organic compounds. Molecularly imprinted artificial recognition membranes have been used for imprinting the analytes of interest. At first the designed sensor array was used for continuous online surveillance and selective quantification of terpenes emanated from species of Lamiaceae family, i.e., peppermint (Mentha × piperita)and basil (Ocimum Basilicum). In terms of monitoring freshness, appreciable reproducibility in emanation patterns comparable to GC-MS analysis was attained with a limit of detection below 70 ppm. Hence, its competency to be explored jointly with pattern recognition tools, i.e., PCA, PLS and ANN. Furthermore, an e-nose with MIP coated biomimetic sensitive layers for comparative study of emanating terpenes of fresh and dried: rosemary (Rosmarinus Officinalis L.), basil (Ocimum Basilicum) and sage (Salvia Officinalis) was made. The optimal e-nose parameters: layer heights, sensitivity <20 ppm of analytes, selectivity at 50 ppm of terpenes, repeatability and reproducibility were systematically achieved. Linearity in reversible responses over a concentration range of ≤ 20-200 ppm has been observed. Isomers, α-pinene and β-pinene can be significantly differentiated by the sensor system. Sensitive and selective properties of e-nose for sensor effects (20–1,200 Hz) have been established which distinguish fresh herbs from dried. The sensor data was analyzed for pattern recognition via PCA and ANN and corroborated with GC-MS results which revealed a similar trend. Moreover, the limit of detection to ≤ 20 ppm and shelf-life of herbs to few days have been examined via designed e-nose. In addition, an ethyl acetate MIP screening strategy has been successfully developed for its chemical sensing. Six MIPs with different monomer compositions of VP, PS and DVB were prepared and tested. Polymer B with monomers ratio (VP: PS: DVB, 1:2:7) was observed as most favorable sensing material for ethyl acetate. Sensitivity and selectivity from a broad range of concentration 25-3000ppm of ethyl acetate was achieved. Cross selectivity of this MIP at 250-750 ppm against 1-propanol was observed to be quite low, i.e., ≤ 1Hz. Finally a tetra-electrode QCM sensor array has been designed. Its fabrication was done through optimizing electrodes size, geometry and with calibration of heating coil. This novel strategy was used for real-time differential mass sensing of terpenes emitted from thyme plant. Patterns of emanating terpenes observed from tetra-electrode QCM array and GC-MS were comparable. Such QCM sensors arrays in practice can explore sensitive and selective concerts for a variety of analytes in different phase’s ranging from bio- micro to macromolecules, e.g., in DNA sensing, monitoring VOCs of plants, composting and degradation process, estimating quality , shelf-life and freshness of food products and in various industries

Développement d'architectures innovantes associant capteurs acoustiques et matériaux polymères à empreintes moléculaires pour la détection de biomarqueurs de cancer

Colorectal cancer statistics in France and all over the world demonstrate theneed for fast, sensitive and specific technological platforms development for cancerdiagnosis. A rapid diagnosis will improve the patients’ health status and reduce the resultswaiting time which could be a great stress factor. Biomarkers analysis in blood, urine andother body fluids is recognized as one of the applied methods for early cancer detection. Inframe of this project, urinary nucleosides have been identified as colorectal cancerbiomarkers. Funded by the National Research Agency (ANR), through the cancer sensorproject (TECSAN program), this thesis was carried out in IMS laboratory. Hence, a colorectalcancer biomarkers detection and monitoring technological solution has been proposed. Inour detection strategy, Molecularly Imprinted Polymers (MIP) has been identified asbiomarker recognition element. The MIP layer has been associated to Love Wave acoustictransducer. This biosensor will sense the identified colorectal cancer nucleosides.Les chiffres des statistiques du cancer colorectal en France et dans le mondemontrent la nécessité de développement de plateformes technologiques plus rapides,sensibles et spécifiques pour assurer le diagnostic du cancer. Un diagnostic rapide va ainsiaider à améliorer l’état de santé et réduire le temps d’attente des résultats qui peut être ungrand facteur de stress pour les patients. L’analyse des biomarqueurs dans le sang, lesurines et autres fluides corporels est l’une des méthodes appliquées pour la détectionprécoce de la maladie. Dans le cadre de ce projet des nucléosides urinaires ont été identifiéscomme biomarqueurs pour le cancer colorectal. Financée par l’Agence Nationale de laRecherche (ANR), à travers le projet CancerSensor (programme TECSAN), cette thèse s’estdéroulée au sein de l’équipe MDA (Microsystèmes de Détection Acoustique) du laboratoireIMS. Dans le cadre de ce projet, nous avons proposé une solution technologique dedétection et de suivi de biomarqueurs du cancer colorectal. Notre choix de la stratégie dedétection s’est porté sur les polymères à empreintes moléculaires comme élément dereconnaissance des biomarqueurs. Celui-ci sera associé à un transducteur acoustique àondes de Love mis au point lors de travaux précédents au sein de l’équipe MDA. Lebiocapteur ainsi développé va cibler les nucléosides mis en évidence pour le cancercolorectal

Detection, Control and Contamination of Mycotoxins

The objective of this collection is to illustrate the most recent research on the development of novel and/or rapid methods for mycotoxin determination, and to propose new strategies for monitoring and/or reducing mycotoxin contamination. Innovative sample preparation techniques or protocols and the possibility of multiclass mycotoxin detection will be very positively considered for possible inclusion in this Special Issue. Both methods based on (bio)sensors and chromatography with various detectors (including mass spectrometry) are welcome. Applications of already published methods on new matrices without any modification will not be accepted. However, extensive studies and monitoring on the spread of contamination through the food production chain could be of interest for this collection

Development of a novel high resolution and high throughput biosensing technology based on a Monolithic High Fundamental Frequency Quartz Crystal Microbalance (MHFF-QCM). Validation in food control

Tesis por compendio[ES] La sociedad actual demanda un mayor control en la seguridad y calidad de los alimentos que se consumen. Esta preocupación se ve reflejada en los diferentes planes estatales y europeos de investigación científica, los cuales, plantean la necesidad de innovar y desarrollar nuevas técnicas analíticas que cubran los requerimientos actuales. En el presente documento se aborda el problema de la presencia de residuos químicos en la miel. El origen de los mismos se debe, fundamentalmente, a los tramientos veterinarios con los que se tratan enfermedades y parásitos en las abejas, y a los tratamientos agrícolas con los que las abejas se ponen en contacto cuando recolectan el néctar en cultivos próximos a las colmenas. La Agencia Europea de Seguridad Alimentaria (EFSA) confirma esta realidad al notificar numerosas alertas sanitarias en la miel. En los últimos años, los métodos de análisis basados en inmunosensores piezoeléctricos se han posicionado como la base de una técnica de cribado muy prometedora, la cual puede ser empleada como técnica complementaria a las clásicas de cromatografía, gracias a su sencillez, rapidez y bajo coste. La tecnología de resonadores High-Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) combina la detección directa en tiempo real, alta sensibilidad y selectividad con un fácil manejo y coste reducido en comparación con otras técnicas. Además, está tecnología permite aumentar el rendimiento del análisis mediante el diseño de arrays de resonadores en un mismo sustrato (Monolithic HFF-QCMD). En este documento se presenta el diseño de un array de 24 sensores HFF-QCMD, junto con un cartucho de micro-fluídica que traza diversos microcanales sobre los diferentes elementos sensores, a los que hace llegar la muestra de miel diluida a analizar. El cartucho actúa también como interfaz para realizar la conexión entre el array de resonadores y el instrumento de caracterización de los mismos. Para obtener el máximo partido del array diseñado, se desarrolla un método de medida robusto y fiable que permite elevar la tasa de adquisición de datos para facilitar la toma de registros eléctricos de un elevado número de resonadores de forma simultánea, e incluso en varios armónicos del modo fundamental de resonancia. La gran sensibilidad de la tecnología HFF-QCMD a los eventos bioquímicos a caracterizar se extiende también a otro tipo eventos externos, como son los cambios de temperatura o presión, lo que es necesario minimizar con el fin de reducir el impacto que estas perturbaciones no deseadas provocan en la estabilidad y fiabilidad de la medida. Con este fin, se desarrolla un algoritmo de procesado de señal basado en la Discrete Transform Wavelet (DTW). Finalmente, todos los desarrollos tecnológicos realizados se validan mediante la implementación de un inmunoensayo para la detección simultánea, en muestras de mieles reales, de residuos químicos de naturaleza química muy diferente, a saber, el fungicida tiabendazol y el antibiótico sulfatiazol.[CA] La societat actual demanda un major control en la seguretat i qualitat dels aliments que es consumeixen. Aquesta preocupació es veu reflectida en els diferents plans estatals i europeus d'investigació científica, els quals, plantegen la necessitat d'innovar i desenvolupar noves tècniques analítiques que cobrisquen els requeriments actuals. En el present document s'aborda el problema de la presència de residus químics en la mel. L'origen dels mateixos es deu, fonamentalment, als tractaments veterinaris amb els quals es tracten malalties i paràsits en les abelles, i als tractaments agrícoles amb els quals les abelles es posen en contacte quan recol·lecten el nèctar en cultius pròxims als ruscos. L'Agència Europea de Seguretat Alimentària (EFSA) confirma aquesta realitat notificant nombroses alertes sanitàries en la mel. En els últims anys, els mètodes d'anàlisis basades en immunosensors piezoelèctrics s'han posicionat com la base d'una tècnica de garbellat molt prometedora, la qual pot ser emprada com a tècnica complementària a les clàssiques de cromatografia, gràcies a la seua senzillesa, rapidesa i baix cost. La tecnologia de ressonadors High-Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) combina la detecció directa en temps real, alta sensibilitat i selectivitat amb un fàcil maneig i cost reduït en comparació amb altres tècniques. A més, està tecnologia permet augmentar el rendiment del anàlisi mitjançant el disseny d'arrays de ressonadors en un mateix substrat (Monolithic HFF-QCMD). En aquest document es presenta el disseny d'un array de 24 sensors HFF-QCMD, juntament amb un cartutx de microfluídica que estableix diversos microcanals sobre els diferents elements sensors, als quals fa arribar la mostra de mel diluïda a analitzar. El cartutx actua també com a interfície per a realitzar la connexió entre l'array de ressonadors i l'instrument de caracterització d'aquests. Per a traure el màxim partit a l'array dissenyat, es desenvolupa un mètode de mesura robust i fiable que permet elevar la taxa d'adquisició de dades per a facilitar la presa de registres elèctrics d'un elevat nombre de ressonadors de manera simultània, i fins i tot en diversos harmònics del mode fonamental de ressonància. La gran sensibilitat de la tecnologia HFF-QCMD als esdeveniments bioquímics a caracteritzar s'estén també a un altre tipus esdeveniments externs, com són els canvis de temperatura o pressió, la qual cosa és necessari minimitzar amb la finalitat de reduir l'impacte que aquestes pertorbacions no desitjades provoquen en l'estabilitat i fiabilitat de la mesura. A aquest efecte, es desenvolupa un algorisme de processament de senyal basat en la Discrete Transform Wavelet (DTW). Finalment, tots els desenvolupaments tecnològics realitzats es validen mitjançant la implementació d'un immunoassaig per a la detecció simultània, en mostres de mel reals, de residus químics de naturalesa química molt diferent, a saber, el fungicida tiabendazol i l'antibiòtic sulfatiazol.[EN] Currently, society demands greater control over the safety and quality of the food consumed. This concern is reflected in the different states and European plans for scientific research, which establish the necessity to innovate and develop new analytical techniques that meet current requirements. This document addresses the problem of the presence of chemical residues in honey. Its origin is fundamentally due to the veterinary treatments against diseases and parasites in bees, and also to the agricultural treatments with which the bees come into contact when they collect the nectar in crops close to the hives. The European Food Safety Agency (EFSA) confirms this reality by notifying numerous health alerts in honey. In recent years, analysis methods based on piezoelectric immunosensors have been positioned as the basis of a very promising screening technique, which can be used as a complementary technique to the classic chromatography, thanks to its simplicity, speed and low cost. High-Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) resonator technology combines direct real-time detection, high sensitivity and selectivity with easy handling and low cost compared to other techniques. In addition, this technology allows increasing the performance of the analysis through the design of resonator arrays on the same substrate (Monolithic HFF-QCMD). This document presents the design of an array of 24 HFF-QCMD sensors, together with a microfluidic cartridge that establish various microchannels on the different sensor elements, to provide them the diluted honey sample to be analyzed. The cartridge also acts as an interface to make the connection between the array of resonators and the characterization instrument. To get the most out of the designed array, a robust and reliable measurement method has been developed that allows increasing the data acquisition rate to facilitate electrical parameters readout from a high number of resonators simultaneously, and even in several harmonics of the fundamental resonance mode. The great sensitivity of the HFF-QCMD technology to the biochemical events to be characterized also is extended to other types of external events, such as changes in temperature or pressure, which must be minimized in order to reduce the impact that these unwanted disturbances cause in the stability and reliability of the measurement. To this end, a signal processing algorithm based on the Discrete Transform Wavelet (DTW) is developed. Finally, all the technological developments carried out are validated through the implementation of an immunoassay for the simultaneous detection, in real honey samples, of chemical residues of very different chemical nature, namely, the fungicide thiabendazole and the antibiotic sulfathiazole.The authors would also like to thank Jorge Martínez from the Laboratory of High Frequency Circuits (LCAF) of the Universitat Politècnica de València (UPV) for assistance with profilometry, and Manuel Planes, José Luis Moya, Mercedes Tabernero, Alicia Nuez and Joaquin Fayos from the Electron Microscopy Services of the UPV for helping with the AFM, and SEM measurements. M.Calero is the recipient of the doctoral fellowship BES-2017-080246 from the Spanish Ministry of Economy, Industry and Competitiveness (Madrid, Spain). This research was funded by Spanish Ministry of Economy and Competitiveness with FEDER funds (AGL 2016-77702-R) and European Commission Horizon 2020 Programme (Grant Agreement number H2020-FETOPEN-2016-2017/737212-CATCH-U-DNA - Capturing non-Amplified Tumor Circulating DNA with Ultrasound Hydrodynamics) for which the authors are grateful. Román Fernández is with the Center for Research and Innovation in Bioengineering (Ci2B), Universitat Politècnica de València, València, Spain and with Advanced Wave Sensors S.L., Paterna, València, Spain. (e-mail: [email protected]); Yolanda Jiménez, Antonio Arnau and María Calero are with the Center for Research and Innovation in Bioengineering (Ci2B), Universitat Politècnica de València, València, Spain; Ilya Reiviakine is with Advanced Wave Sensors S.L., Paterna, Valencia, Spain and with the Department of Bioengineering, University of Washington, Seattle, WA, 98150 USA; María Isabel Rocha-Gaso and José Vicente García are with Advanced Wave Sensors S.L., Paterna, València, Spain.Calero Alcarria, MDS. (2022). Development of a novel high resolution and high throughput biosensing technology based on a Monolithic High Fundamental Frequency Quartz Crystal Microbalance (MHFF-QCM). Validation in food control [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182652TESISCompendi

Label-free and Multi-parametric Monitoring of Cell-based Assays with Substrate-embedded Sensors

Various approaches have been pursued on the basis of electrochemical or piezoelectric transducers, particularly of the quartz crystal microbalance (QCM), to monitor non-invasively and in real-time cellular states and reactions with substrate-embedded sensors. On the one hand, these comprised the technical development of piezoelectric sensors with multiple read-out spots and the integration of additional non-invasive (electro- and optochemical) sensor technologies on the QCM surface. On the other hand, a variety of studies and cell-based assays (CBAs) have been performed in order to test the sensor performances and to gain a deeper understanding of the sensors’ readout parameters with respect to their information content about the biophysical properties and the metabolic behavior of cells. Fig. 7–1 presents an overview of the different projects on the basis of QCM sensor disks presented in this thesis. In the first project (Fig. 7–1 A) a novel electrode layout was designed on the basis of commercial 5 MHz AT-cut quartz disks to implement two independent readout spots on the QCM surface. This also comprised the construction of new measurement chambers for the electrical actuation and sensing of quartz oscillations. These two-electrode QCM sensors (2ElQ) are also referred to as multichannel QCM (MQCM). The developed MQCM sensor spots on one quartz disk exhibited a strong interference, even though they were operated sequentially, which is in contrast to the results of previous theoretical calculations. The resonances could be successfully decoupled by coating half of the quartz surface and one sensor spot, respectively, with a thin and rigid film of photoresist. This quartz loading with mass caused a shift in the resonance spectra of the coated resonator to lower frequencies and efficient decoupling. The operation of the decoupled MQCM sensors demonstrated both, a sensitive and equal change in the oscillation characteristics of the two resonators upon loading of the quartz with medium. The Q-factor was not significantly different for the two resonators, qualifying the MQCM for its application in CBAs. Building on the preceding development of the double-electrode quartz disks, a novel electrode layout has been realized at the sensor surface, which enables the complementary electrochemical (impedance spectroscopical) characterization of the substrate-liquid interface in addition to its mechanical characterization by the piezoelectric transducers (Fig. 7–1 B). This layout was achieved by removing a small area of the insulating photoresist on the coated electrode in the photolithographic process. By this, a coplanar electrode arrangement of a small working electrode and a bigger counter electrode was created. This sensor combination on the basis of the MQCM is an improvement of the so-called 2nd generation QCM to what we call the 3rd generation QCM, and which is also referred to as QCM-ECIS. Various electrode layouts, varying in size and number of the working electrode(s), were fundamentally characterized microscopically and by profilometry regarding the geometrical properties and by means of impedance spectroscopy with respect to the sensing performances in QCM- and ECIS-mode. An optimal electrode layout was identified and defined as standard for subsequent applications in CBAs. In both QCM- and ECIS studies of cell-covered sensor surfaces significant changes in the characteristic sensing parameters with respect to the cell-free electrodes are measurable. In addition to the measurement of absolute signal changes, the transducer technologies of QCM and ECIS also enable to monitor the kinetic changes of the readout parameters with high temporal resolution. This allows to use the dual sensors for monitoring and analyzing the states of adherent cell cultures in any kind of assay, label-free, non-invasively, and in real-time. Mechanical (QCM-mode) and the dielectric (ECIS-mode) characteristics of cell adhesion were simultaneously measured for two different cell lines (MDCK II and NRK), with high reproducibility for each. The total and kinetic parameter changes in both sensing modes distinguished clearly and were specific for the cell lines under test. The signals from both QCM-mode and ECIS-mode recordings also reported on significant impacts of the presence/ absence of bivalent cations (Ca2+, Mg2+) on the attachment and spreading kinetics and behavior of MDCK II cells. Aside from cell adhesion studies, the cytomechanical and cell morphological reactions towards various stimuli were monitored and analyzed by QCM-ECIS in a multitude of cellular assays: systematic softening and stiffening of cells (using agents for disassembling the actin cytoskeleton and cross-linking protein structures, respectively), intracellular stimulation (using a second messenger analogue), as well as electrical manipulation (electroporation (ELPO) and wounding) of cell layers (applying invasive voltage pulses). The applicability of electrical actuation and the subsequent non-invasive, time-resolved, and dual sensing with the electrodes of the QCM-ECIS substrates has been successfully demonstrated. The monitoring of CBAs with the dual QCM-ECIS sensor chips developed in this thesis provides not only a multiplication of the information gain due to the complementarity of QCM and ECIS readout parameters. The simultaneous, time-resolved measurements also enable the kinetic correlation of the sensor signals in novel 2 D and 3 D diagrams, which offers the hitherto unprecedented opportunity for a more detailed view and analysis of the coherence or consecutiveness of mechanical and morphological/ dielectric changes of a cell layer under study. A third research project focused on the combination of optical-chemical sensors (OCS) with the piezoelectric (QCM) sensor technology. For this purpose, the quartz crystal surface was coated with a polymer film with embedded phosphorescent indicator dye for the target analyte. The luminescence properties were measured by means of fluorescence (phosphorescence) lifetime imaging (FLIM). By using a temperature-sensitive paint (TSP), an increase in temperature on the sensor surface upon high-amplitude oscillations was monitored and imaged this way in one project (Fig. 7–1 C). Based on this experimentally determined local heating on the QCM surface and the thereby generated temperature gradient in the liquid above the resonator, a thermophoretic convection in the fluid has been simulated. Theoretical considerations showed that the convection profile in the measurement vessel counteracts and even largely prevents the sedimentation of cells onto the sensor surface. It is suggested that the effect of thermophoresis is crucial especially in studies of biomolecular interactions on QCM surfaces at elevated shear amplitudes and driving voltages, respectively, which however has not been considered in literature to date. The phosphorescence quenching capability of oxygen was utilized in a second imaging project to monitor and image the local concentration and distribution of oxygen on the growth substrate of cells by means of a so-called pressure(/oxygen)-sensitive paint (PSP) (Fig. 7–1 D). A home-made experimental setup was constructed for sensor calibration and the imaging of subcellular oxygen, consisting of a FLIM setup coupled to an upright microscope and a temperature- and oxygen-controlled calibration and measurement chamber suitable for cellular applications. The cytocompatible sensor films have been characterized under various test conditions (in air, under medium, at different temperatures) regarding their sensitivity and response characteristics to different oxygen partial pressures. The oxygen consumption of cells adherently grown on the sensor film was successfully monitored and imaged by this setup. The time-resolved measurements demonstrated a significantly faster consumption of oxygen of a cell layer stimulated with a respiration chain decoupler compared to an unstimulated control cell layer. Taken together, various technical improvements of piezoelectric sensors (QCM) have been realized (MQCM, QCM-ECIS, ELPO-QCM-ECIS, QCM-OCS), which provide a significant information gain in cell-based applications. The sensors developed enable the high-content screening (HCS) of adherent cell lines in a wide range of assay formats and provide complementary physico-chemical information for obtaining a more complete picture of the state of cells and their reactions in contact to diverse stimuli. All sensor techniques share the characteristics of time-resolved, label-free, and non-invasive monitoring. This allows to disclose and analyze even the kinetics, delayed effects, recoveries, and fluctuations of physicochemical alterations of a studied cell layer, in addition to the absolute parameter changes, which is a valuable improvement compared to classical endpoint assays. The approach of combined, independent sensor systems also provides the novel possibility to bring parameters obtained by the different readout technologies from one cell layer in a temporal correlation, by which new insights into physiological relationships are possible