Theoretical and experimental development of a ZnO-based laterally excited thickness shear mode acoustic wave immunosensor for cancer biomarker detection

The object of this thesis research was to develop and characterize a new type of acoustic biosensor - a ZnO-based laterally excited thickness shear mode (TSM) resonator in a solidly mounted configuration. The first specific aim of the research was to develop the theoretical underpinnings of the acoustic wave propagation in ZnO. Theoretical calculations were carried out by solving the piezoelectrically stiffened Christoffel equation to elucidate the acoustic modes that are excited through lateral excitation of a ZnO stack. A finite element model was developed to confirm the calculations and investigate the electric field orientation and density for various electrode configurations. A proof of concept study was also carried out using a Quartz Crystal Microbalance device to investigate the application of thickness shear mode resonators to cancer biomarker detection in complex media. The results helped to provide a firm foundation for the design of new gravimetric sensors with enhanced capabilities. The second specific aim was to design and fabricate arrays of multiple laterally excited TSM devices and fully characterize their electrical properties. The solidly mounted resonator configuration was developed for the ZnO-based devices through theoretical calculations and experimentation. A functional mirror comprised of W and SiO2 was implemented in development of the TSM resonators. The devices were fabricated and tested for values of interest such as Q, and electromechanical coupling (K2) as well as their ability to operate in liquids. The third specific aim was to investigate the optimal surface chemistry scheme for linking the antibody layer to the ZnO device surface. Crosslinking schemes involving organosilane molecules and a phosphonic acid were compared for immobilizing antibodies to the surface of the ZnO. Results indicate that the thiol-terminated organosilane provides high antibody surface coverage and uniformity and is an excellent candidate for planar ZnO functionalization. The fourth and final specific aim was to investigate the sensitivity of the acoustic immunosensors to potential diagnostic biomarkers. Initial tests were performed in buffer spiked with varying concentrations of the purified target antigen to develop a dose-response curve for the detection of mesothelin-rFc. Subsequent tests were carried out in prostate cancer cell line conditioned medium for the detection of PSA. The results of the experiments establish the operation of the devices in complex media, and indicate that the acoustic sensors are sensitive enough for the detection of biomolecular targets at clinically relevant concentrations.Ph.D.Committee Chair: William D Hunt; Committee Member: Bruno Frazier; Committee Member: Dale Edmondson; Committee Member: Marie Csete; Committee Member: Peter Edmonson; Committee Member: Ruth O'Rega

In-liquid bulk acoustic wave resonators for biosensing applications

Gravimetric sensors based on thin-film bulk acoustic wave (BAW) resonators operating between 1-5 GHz have tremendous potential as biosensors because they are inexpensive, label-free, fast and highly sensitive. The two main challenges in this objective are: the conventional longitudinal mode resonance in $\textit{c}$-axis oriented piezoelectric films suffers from more than 90% damping in liquid; the alternative is the shear mode resonance, with lower damping in liquid but which requires an inclined $\textit{c}$-axis piezoelectric film, a process that is still not fully scalable. In this thesis, seed layers such as AlN with mainly (103) orientations are used to promote the growth of homogeneously inclined $\textit{c}$-axis ZnO (inclination of up to $\sim$45$^{\circ}$) films without significant equipment modifications. Sputtered Al electrodes with controlled roughness are then substituted for the parasitic AlN seed layers to improve the electromechanical performance. At a substrate temperature, T$_{s}$ = 100 $^{\circ}$C, an optimum surface roughness of 9.2 nm yields homogeneously inclined $\textit{c}$-axis ZnO films with angles $\sim$25$^{\circ}$. Solidly mounted resonators (SMRs) operating in a shear mode at $\sim$1.1 GHz with the Al electrodes have resonant quality factors (Q$_{r}$) higher than 150 and effective electromechanical coupling coefficients, k$^{2}$$_{eff}$, of 2.9-3.4%, which are improved from only 2.2% with the AlN seed layers. This shear mode of the ZnO SMRs has mass sensitivities, S$_{m}$ of (4.9 $\pm$ 0:1) kHz$\cdot$cm$^{2}$/ng and temperature coefficients of frequency (TCF) of -(66$\pm$2) ppm/K. Viscosity sensing is carried out with different ethanol-water compositions; the SMRs are functionalised and successfully used in the detection of Rabbit Immunoglobin G. To mitigate the longitudinal mode damping in water, multi-wall carbon nanotube (CNT) forests are grown by chemical vapour deposition (CVD) at 600 $^{\circ}$C using Fe/Al layers on the active area of inclined $\textit{c}$-axis AlN SMRs designed for improved thermal and chemical stability. The dense CNT forest (with 0.5/8 nm Fe/Al) of $\sim$15 μm height provides an acoustic isolation to DI water with only 50-70% drop in the longitudinal mode Q$_{r}$ compared to 99% in SMRs without the CNTs. Mass loading is still detected and demonstrated by detecting bovine serum albumin (BSA) in water whereas with forest heights of $\sim$30 μm and no significant frequency shifts due to mass attachment are observed. With the CNTs the longitudinal mode is shown for the first time to be more sensitive to mass ($\sim$7x) than the shear mode in liquid, highlighting the potential of CNTs for the large scale use of the longitudinal mode for in-liquid sensing

A review of thermal impact of surface acoustic waves on microlitre droplets in medical applications

The surface acoustic waves (SAW) propagate inside the microdroplets resulting in kinetic and thermal impacts. The kinetic drives fluid particles inside the droplet while thermal impact increases the liquid’s temperature. This paper provides a comprehensive review of the research investigations related to internal kinetics and heating inside the microdroplet caused by the acoustic waves. The main factors that affect the kinetics and convection heat transfer are the piezoelectric materials, shape of the interdigital transducer (IDT) and mode of acoustic waves. Internal streaming (kinetic) leads to particle mixing, particle manipulation, cell sorting, cell patterning, cell separation, measuring the concentration of immunoglobulin and so forth. The effect of changing the mode of waves and the shape of IDT on the relevant applications are presented. Internal convection heat transfer is important where heating of the liquid is essential for many applications such as monitoring blood coagulation in the human plasma and an acoustic tweezer for particle trapping. Experimental methods developed by researchers to realise uniform temperature with constant heating and cooling cycles are also discussed. Such methods are widely used in the polymerase chain reaction (PCR) to detect COVID-19 infection. The heating of the droplet can be efficiently controlled by changing the input power and by varying the duty factor

Based acoustic waves microsensor for the detection of bacteria in complex liquid media

Cette thèse s’inscrit dans le cadre d’une cotutelle internationale entre l’Université de Bourgogne Franche-Comté en France et l’Université de Sherbrooke au Canada. Elle porte sur le développement d'un biocapteur miniature pour la détection et la quantification de bactéries dans des milieux liquides complexes. La bactérie visée est l’Escherichia coli (E. coli), régulièrement mise en cause dans des épidémies d'infections alimentaires, et parfois meurtrière. La géométrie du biocapteur consiste en une membrane en arséniure de gallium (GaAs) sur laquelle est déposé un film mince piézoélectrique d’oxyde de zinc (ZnO). L'apport du ZnO structuré en couche mince constitue un réel atout pour atteindre de meilleures performances du transducteur piézoélectrique et consécutivement une meilleure sensibilité de détection. Une paire d'électrodes déposée sur le film de ZnO permet de générer, sous une tension sinusoïdale, des ondes acoustiques se propageant dans le GaAs, à une fréquence donnée. La face arrière de la membrane, quant à elle, est fonctionnalisée avec une monocouche auto-assemblée (SAM) d'alkanethiols et des anticorps contre l’E. coli, conférant la spécificité de la détection. Ainsi, le biocapteur bénéficie à la fois des technologies de microfabrication et de bio-fonctionnalisation du GaAs, déjà validées au sein de l’équipe de recherche, et des propriétés piézoélectriques prometteuses du ZnO, afin d’atteindre potentiellement une détection hautement sensible et spécifique de la bactérie d’intérêt. Le défi consiste à pouvoir détecter et quantifier cette bactérie à de très faibles concentrations dans un échantillon liquide et/ou biologique complexe. Les travaux de recherche ont en partie porté sur les dépôts et caractérisations de couches minces piézoélectriques de ZnO sur des substrats de GaAs. L’effet de l’orientation cristalline du GaAs ainsi que l’utilisation d’une couche intermédiaire de Platine entre le ZnO et le GaAs ont été étudiés par différentes techniques de caractérisation structurale (diffraction des rayons X, spectroscopie Raman, spectrométrie de masse à ionisation secondaire), topographique (microscopie à force atomique), optique (ellipsométrie) et électrique. Après la réalisation des contacts électriques, la membrane en GaAs a été usinée par gravure humide. Une fois fabriqué, le transducteur a été testé en air et en milieu liquide par des mesures électriques, afin de déterminer les fréquences de résonance pour les modes de cisaillement d’épaisseur. Un protocole de bio-fonctionnalisation de surface, validé au sein du laboratoire, a été appliqué à la face arrière du biocapteur pour l’ancrage des SAMs et des anticorps, tout en protégeant la face avant. De plus, les conditions de greffage d’anticorps en termes de concentration utilisée, pH et durée d’incubation, ont été étudiées, afin d’optimiser la capture de bactérie. Par ailleurs, l’impact du pH et de la conductivité de l’échantillon à tester sur la réponse du biocapteur a été déterminé. Les performances du biocapteur ont été évaluées par des tests de détection de la bactérie cible, E. coli, tout en corrélant les mesures électriques avec celles de fluorescence. Des tests de détection ont été réalisés en variant la concentration d’E. coli dans des milieux de complexité croissante. Différents types de contrôles ont été réalisés pour valider les critères de spécificité. En raison de sa petite taille, de son faible coût de fabrication et de sa réponse rapide, le biocapteur proposé pourrait être potentiellement utilisé dans les laboratoires de diagnostic clinique pour la détection d’E. coli.Abstract: This thesis was conducted in the frame of an international collaboration between Université de Bourgogne Franche-Comté in France and Université de Sherbrooke in Canada. It addresses the development of a miniaturized biosensor for the detection and quantification of bacteria in complex liquid media. The targeted bacteria is Escherichia coli (E. coli), regularly implicated in outbreaks of foodborne infections, and sometimes fatal. The adopted geometry of the biosensor consists of a gallium arsenide (GaAs) membrane with a thin layer of piezoelectric zinc oxide (ZnO) on its front side. The contribution of ZnO structured in a thin film is a real asset to achieve better performances of the piezoelectric transducer and consecutively a better sensitivity of the detection. A pair of electrodes deposited on the ZnO film allows the generation of acoustic waves propagating in GaAs under a sinusoidal voltage, at a given frequency. The backside of the membrane is functionalized with a self-assembled monolayer (SAM) of alkanethiols and antibodies against E. coli, providing the specificity of the detection. Thus, the biosensor benefits from the microfabrication and bio-functionalization technologies of GaAs, validated within the research team, and the promising piezoelectric properties of ZnO, to potentially achieve a highly sensitive and specific detection of the bacteria of interest. The challenge is to be able to detect and quantify these bacteria at very low concentrations in a complex liquid and/or biological sample. The research work was partly focused on the deposition and characterization of piezoelectric ZnO thin films on GaAs substrates. The effect of the crystalline orientation of GaAs and the use of a titanium/platinum buffer layer between ZnO and GaAs were studied using different structural (X-ray diffraction, Raman spectroscopy, secondary ionization mass spectrometry), topographic (atomic force microscopy), optical (ellipsometry) and electrical characterizations. After the realization of the electrical contacts on top of the ZnO film, the GaAs membrane was micromachined using chemical wet etching. Once fabricated, the transducer was tested in air and liquid medium by electrical measurements, in order to determine the resonance frequencies for thickness shear mode. A protocol for surface bio-functionalization, validated in the laboratory, was applied to back side of the biosensor for anchoring SAMs and antibodies, while protecting the top side. Furthermore, different conditions of antibody immobilization such as the concentration, pH and incubation time, were tested to optimize the immunocapture of bacteria. In addition, the impact of the pH and the conductivity of the solution to be tested on the response of the biosensor has been determined. The performance of the biosensor was evaluated by detection tests of the targeted bacteria, E. coli, while correlating electrical measurements with fluorescence microscopy. Detection tests were completed by varying the concentration of E. coli in environments of increasing complexity. Various types of controls were performed to validate the specificity criteria. Thanks to its small size, low cost of fabrication and rapid response, the proposed biosensor has the potential of being applied in clinical diagnostic laboratories for the detection of E. coli

Bulk and Surface Acoustic Wave Biosensors for Milk Analysis

Milk and dairy products are common foods and, therefore, are subject to regular controls. Such controls cover both the identification and quantification of specific components and the determination of physical parameters. Components include the usual milk ingredients, mainly carbohydrates, proteins, and fat, and any impurities that may be present. The latter range from small molecules, such as drug residues, to large molecules, e.g., protein-based toxins, to pathogenic microorganisms. Physical parameters of interest include viscosity as an indicator of milk gelation. Bulk and surface acoustic wave sensors, such as quartz crystal microbalance (QCM) and surface acoustic wave (SAW) devices, can principally be used for both types of analysis, with the actual application mainly depending on the device coating and the test format. This review summarizes the achievements of acoustic sensor devices used for milk analysis applications, including the determination of physical liquid parameters and the detection of low- and high-molecular-weight analytes and microorganisms. It is shown how the various requirements resulting from the respective analytes and the complex sample matrix are addressed, and to what extent the analytical demands, e.g., with regard to legal limits, are met