Detection of Angiogenic Growth Factor by Microcantilever Biosensors

2009/2010To reach new and relevant insights in biomolecular sciences, new tools for fine and precise measurement are needed. Nowadays advances in the field of micro-electro-mechanical systems (MEMS) offer unique opportunities in the design of ultrasensitive analytical devices to support the molecular sensing investigations. Among them Microcantilever (MC) biosensors are label-free platforms that combine a biologically sensitive with a physical transducer in order to selectively and quantitatively detect the presence of specific compounds in a given external environment. Since they can be operated either as nanomechanical resonator or as surface stress sensor, MCs - activated with DNA probes or antibodies for molecular recognition - enable the measurement of mass with extraordinary sensitivity. In particular, the development of mass detector biosensor based on MC systems would permit to shift from qualitative data to quantitative measurements of key molecules involved in physiological processes. This can lead crucial informations to characterize complex mechanisms such as angiogenesis and tumor progression and to the quantification of small amounts of cancer markers, such as Angiopoietin-1 (ANG-1), and their modulation during the early stages of tumor development.XXIII Ciclo197

Monitoring CO Concentration in Fuel Cells Using Microcantilever Sensors

Estimating the concentration of gases including carbon monoxide (CO) in the hydrogen fuel exiting the reformer and entering the fuel cell is imperative. A high concentration of CO can cause fuel-cell catalyst poisoning, which permanently destroys the cell. Current practices call for utilizing expensive and bulky spectral analyzers to achieve this task. In addition to their high cost, these methodologies, undoubtedly, hinder the portability and self-containment of the cell. To overcome these problems and achieve the desired objectives of a portable, self-contained, and real-time measurement module, this thesis presents and experimentally investigates a new enabling technology based on utilizing an array of microcantilever sensors to detect minute concentrations of CO in the fuel cell. Results of this study indicate that microcantilevers can be spin coated with homogenous layers of copper-exchanged Y zeolite (CuY). This zeolite is capable of adsorbing CO over a range pressures and fuel cell operating temperatures. As a result of this adsorption, the sensor experiences a shift in its resonance frequency, which can be measured and related to the concentration of CO. It is determined that maximum adsorption capacity of the sensor occurs at 40 oC using CuY zeolite that is loaded with 10 wt% Cu. Furthermore, experimental findings indicate that the sensitivity of the sensor increases as the number of zeolite layers is increased up to a certain threshold (4 layers). Beyond this threshold, adding more layers will only result in a less sensitive sensor. In the experiments described in this thesis, a maximum repeatable shift of 275 Hz in the first modal frequency of the microcantilevers is measured. Ultimately, such frequency shifts can be iii related to the concentration of CO in the gas mixture, allowing closed-loop, real-time control and diagnosis of the flow of gases into and out of the fuel cell. This can help avoid fuel-cell starvation and prevent catastrophic deactivation of the necessary fuel cell catalyst

Microelectromechanical Systems and Devices

The advances of microelectromechanical systems (MEMS) and devices have been instrumental in the demonstration of new devices and applications, and even in the creation of new fields of research and development: bioMEMS, actuators, microfluidic devices, RF and optical MEMS. Experience indicates a need for MEMS book covering these materials as well as the most important process steps in bulk micro-machining and modeling. We are very pleased to present this book that contains 18 chapters, written by the experts in the field of MEMS. These chapters are groups into four broad sections of BioMEMS Devices, MEMS characterization and micromachining, RF and Optical MEMS, and MEMS based Actuators. The book starts with the emerging field of bioMEMS, including MEMS coil for retinal prostheses, DNA extraction by micro/bio-fluidics devices and acoustic biosensors. MEMS characterization, micromachining, macromodels, RF and Optical MEMS switches are discussed in next sections. The book concludes with the emphasis on MEMS based actuators

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

Microcantilever-based sensing arrays for evaluation of biomolecular interactions

The controlled immobilization on a surface of biomolecules used as recognition elements is of fundamental importance in order to realize highly specific and sensible biosensors. Microcantilevers (MC) are nanomechanical sensors, which can be used as label free micro-sized mechanical transducers. MC resonant frequency is sensitively modified upon molecules adsorption, demonstrating an impressive mass resolution. A widely used approach for the immobilization of biorecognition elements on silicon substrates consists in the deposition of 3-aminopropyl-triethoxysilane (APTES) followed by the incubation with glutaraldehyde (GA) as a crosslinking agent. However, these derivatization processes produce a variable chemical functionalization because of the spontaneous polymerization of GA in aqueous solutions. With the aim of producing a more reliable chemical functionalization for protein immobilization, the deposition of a thin film of APTES by self-assembly followed by the modification of its amino groups into carboxyl groups by incubating in succinic anhydride (SA) is proposed. Moreover, the activation of these terminal carboxyl groups were performed by using the EDC/s-NHS protocol in order to enhance their reactivity toward primary amine groups present on biomolecules surface. This method was characterized from a physico-chemical point of view by means of compositional and morphological surface analysis. Moreover, data acquired after the application of this functionalization to a MC-based system showed a highly reproducible deposition of APTES/SA when compared to APTES/GA deposition process. APTES/SA derivatized MC arrays were then incubated with biomolecules for the study of its protein binding capability: the quantification of the grafted biomolecules was performed from the gravimetric data and compared with a theoretical surface density calculated through a molecular modeling tool, providing information about the orientation of the proteins tethered to the surface. In order to avoid or reduce non-specific protein interactions, Bovine Serum Albumin and ethanolamine were considered for their blocking capability. Finally, the detection of the envelope glycoprotein domain III of the Dengue virus type 1 based on immune-specific recognition through the DV32.6 antibody was performed, providing a stoichiometry ratio for the DIII-DV1/DV32.6 interaction. Currently, no cure or vaccine are available; thus, a better understanding of the interactions between the viruses and specific antibodies is expected to provide fundamental information for the development of a vaccine

Novel Cantilever Sensor for Antibody-based and hlyA gene-based Sensitive Detection of Foodborne Pathogen: Listeria monocytogenes

The objectives of this research can be broadly categorized as: 1) Cantilever sensor design and characteristics, 2) Improved surface chemistry for immobilization of recognition molecules and 3) Sensing of contaminants in food and cell-culture matrix.A novel asymmetrically anchored piezoelectric millimeter-sized cantilever (aPEMC) design was developed. The new cantilever design is simpler and has lesser fabrication variables that improved the reproducibility of these devices. The sensor design was corroborated and characterized using finite element modeling and these were shown to be highly sensitive (~1 fg/Hz) via molecular chemisorption studies. The importance of the binding strength between the sensor and the added mass was shown to govern the type of resonance frequency change exhibited by the cantilever sensors and the high-order resonance oscillation modes were characterized by comparing responses to that quartz crystal microbalance (QCM) and finite element modeling.A novel and dry method for grafting reactive amine groups on polyurethane surfaces using pulsed-plasma generation of ammonia gas was demonstrated. Grafting of amine groups was corroborated by FTIR studies and SEM micrographs in addition to the cantilever sensor responses to protein immobilization. A method using tris(2-carboxyethyl)phosphine (TCEP) to reduce the disulfide bridges in antibody molecules, without affecting the antigen binding activity, to expose their native thiol groups for immobilization of gold surfaces was developed. The half antibody fragments were shown to improve the detection sensitivity of QCM biosensors without loss of selectivity.Piezoelectric millimeter-sized cantilever (PEMC) sensors were used to demonstrate detection of cell-culture mycoplasmas in buffer and cell-culture matrix at 103 CFU/mL. The detection responses were confirmed by using a second antibody binding step, much like ELISA sandwich format. aPEMC sensors were used to show detection of foodborne pathogen, Listeria monocytogenes (LM), in buffer and milk at concentration of 103/mL. The detection sensitivity was limited by commercially available low-avidity antibody. The single copy, virulence hlyA gene of LM was used to design a DNA probe that was used to detect genomic DNA extracted from LM in the presence of ~104 times higher non-target genomic DNA. Detection of genomic DNA equivalent to 7×102 LM was achieved within ~90 min.Ph.D., Chemical Engineering -- Drexel University, 201

Piezoelectric microcantilever serum protein detector

The development of a serum protein detector will provide opportunities for better screening of at‐risk cancer patients, tighter surveillance of disease recurrence and better monitoring of treatment. An integrated system that can process clinical samples for a number of different types of biomarkers would be a useful tool in the early detection of cancer. Also, screening iomarkers such as antibodies in serum would provide clinicians with information regarding the patient’s response to treatment. Therefore, the goal of this study is to develop a sensor which can be used for rapid, all‐electrical, real‐time, label‐fee, in‐situ, specific quantification of cancer markers, e.g., human epidermal receptor 2 (Her2)or antibodies, in serum. To achieve this end, piezoelectric microcantilever sensors (PEMS) were constructed using an 8 μm thick lead magnesium niobate‐lead titanate (PMN‐PT) freestanding film as the piezoelectric layer. The desired limit of detection is on the order of pg/mL. In order to achieve this goal the higher frequency lateral extension modes were used. Also, as the driving and sensing of the PEMS is electrical, the PEMS must be insulated in a manner that allows it to function in aqueous solutions. The insulation layer must also be compatible with standardized bioconjugation techniques. Finally, detection of both cancer antigens and antibodies in serum was carried out, and the results were compared to a standard commercialized protocol. PEMS have demonstrated the capability of detecting Her2 at a concentration of 5 pg/mL in diluted human serum (1:40) in less than 1 hour. The approach can be easily translated into the clinical setting because the sensitivity is more than sufficient for monitoring prognosis of breast cancer patients. In addition to Her2 detection, antibodies in serum were assayed in order to demonstrate the feasibility of monitoring the immune response for antibody‐dependent cellular cytotoxicity (ADCC) in patients on antibody therapies such as Herceptin and Cetuximab. The PEMS displayed a limit of detection of 100 fg/mL, which was 100 times lower than the current methods of protein detection in serum, such as ELISA. Furthermore, the sensitivity of the PEMS device allows it to be capable of determining the dissociation constant, Kd, of selective receptors such as antibodies. Using the dose response trials of Her2, Kd has been deduced for H3 scFv, and Herceptin, a commercial antibody specific for Her2.Ph.D., Materials Engineering -- Drexel University, 200

Flexible ZnO thin film-based surface acoustic wave devices for environmental and biomedical sensing applications

Flexible ZnO thin film on aluminium foil-based SAW devices have been investigated for the first time as sensors for temperature, UV light, and humidity as well as breath and apnoea detection, and these devices were performing sensing while they were placed in flat and bending (curved) positions. Flexible SAW devices offer a promising technology of low cost, highly sensitive and bendable sensors. They also exhibit high potential for wearable, point of care and microfluidics and lab-on-chips applications. The ZnO thin film was deposited on the aluminium foil and ZnO nanorods were grown on the surface of selected samples. The SAW sensors were fabricated by patterning Au/Cr IDTs with various wavelengths. Film and nanorods possessed the preferred structure and piezoelectric properties. Lamb modes were identified, and they were in a good agreement with the FEA results. The maximum value of TCF was -773 ppm/K which is among the highest values mentioned in the literature. The sensors showed excellent linearity and repeatability during temperature cycling test. The maximum value of sensitivity to UV light was 63 ppm (mW/cm2)-1. ZnO nanorods enhanced the sensitivity by 1.76 times. The sensors showed excellent repeatability and reliability during UV light cycling in flat, bent-up and bent-down positions. The maximum values of sensitivity to humidity were 47.7 kHz at 90%RH for nanorodenhanced device and the maximum frequency shift was -57 kHz. The sensors exhibited good repeatability in response to humidity cycling. Besides, the devices exhibited an excellent response, sensitivity, and reliability for various breath patterns (e.g., healthy breathing, apnoea, slow and fast breathing)