CMOS integration of AlN based piezoelectric microcantilevers

El gran crecimiento de los sistemas MEMS (Micro Electro Mechanical Systems) así como su presencia en la mayoría de los dispositivos que usamos diariamente despertó nuestro interés. Paralelamente, la tecnología CMOS (Complementary Metal Oxide Semiconductor) es la tecnología más utilizada para la fabricación de circuitos integrados. Además de ventajas relacionadas con el funcionamiento electrónico del dispositivo final, la integración de sistemas MEMS en la tecnología CMOS reduce significantemente los costes de fabricación. Algunos de los dispositivos MEMS con mayor variedad de aplicaciones son los microflejes. Estos dispositivos pueden ser utilizados para la extracción de energía, en microscopios de fuerza atómica o en sensores, como por ejemplo, para biodetección. Los materiales piezoeléctricos más comúnmente utilizados en aplicaciones MEMS se sintetizan a altas temperaturas y por lo tanto no son compatibles con la tecnología CMOS. En nuestro caso hemos usado nitruro de alumino (AlN), que se deposita a temperatura ambiente y es compatible con la tecnología CMOS. Además, es biocompatible, y por tanto podría formar parte de un dispositivo que actúe como biosensor. A lo largo de esta tesis hemos prestado especial atención en desarrollar un proceso de fabricación rápido, reproducible y de bajo coste. Para ello, todos los pasos de fabricación han sido minuciosamente optimizados. Los parámetros de sputtering para depositar el AlN, las distintas técnicas y recetas de ataque, los materiales que actúan como electrodos o las capas sacrificiales para liberar los flejes son algunos de los factores clave estudiados en este trabajo. Una vez que la fabricación de los microflejes de AlN ha sido optimizada, fueron medidos para caracterizar sus propiedades piezoeléctricas y finalmente verificar positivamente su viabilidad como dispositivos piezoeléctricos. ABSTRACT The huge growth of MEMS (Micro Electro Mechanical Systems) as well as their presence in most of our daily used devices aroused our interest on them. At the same time, CMOS (Complementary Metal Oxide Semiconductor) technology is the most popular technology for integrated circuits. In addition to advantages related with the electronics operation of the final device, the integration of MEMS with CMOS technology reduces the manufacturing costs significantly. Some of the MEMS devices with a wider variety of applications are the microcantilevers. These devices can be used for energy harvesting, in an atomic force microscopes or as sensors, as for example, for biodetection. Most of the piezoelectric materials used for these MEMS applications are synthesized at high temperature and consequently are not compatible with CMOS technology. In our case we have used aluminum nitride (AlN), which is deposited at room temperature and hence fully compatible with CMOS technology. Otherwise, it is biocompatible and and can be used to compose a biosensing device. During this thesis work we have specially focused our attention in developing a high throughput, reproducible and low cost fabrication process. All the manufacturing process steps of have been thoroughly optimized in order to achieve this goal. Sputtering parameters to synthesize AlN, different techniques and etching recipes, electrode material and sacrificial layers are some of the key factors studied in this work to develop the manufacturing process. Once the AlN microcantilevers fabrication was optimized, they were measured to characterize their piezoelectric properties and to successfully check their viability as piezoelectric devices.

Compilador de consultas tipo SQL para sistema de procesamiento masivo de eventos CEP

El paradigma de procesamiento de eventos CEP plantea la solución al reto del análisis de grandes cantidades de datos en tiempo real, como por ejemplo, monitorización de los valores de bolsa o el estado del tráfico de carreteras. En este paradigma los eventos recibidos deben procesarse sin almacenarse debido a que el volumen de datos es demasiado elevado y a las necesidades de baja latencia. Para ello se utilizan sistemas distribuidos con una alta escalabilidad, elevado throughput y baja latencia. Este tipo de sistemas son usualmente complejos y el tiempo de aprendizaje requerido para su uso es elevado. Sin embargo, muchos de estos sistemas carecen de un lenguaje declarativo de consultas en el que expresar la computación que se desea realizar sobre los eventos recibidos. En este trabajo se ha desarrollado un lenguaje declarativo de consultas similar a SQL y un compilador que realiza la traducción de este lenguaje al lenguaje nativo del sistema de procesamiento masivo de eventos. El lenguaje desarrollado en este trabajo es similar a SQL, con el que se encuentran familiarizados un gran número de desarrolladores y por tanto aprender este lenguaje no supondría un gran esfuerzo. Así el uso de este lenguaje logra reducir los errores en ejecución de la consulta desplegada sobre el sistema distribuido al tiempo que se abstrae al programador de los detalles de este sistema.---ABSTRACT---The complex event processing paradigm CEP has become the solution for high volume data analytics which demand scalability, high throughput, and low latency. Examples of applications which use this paradigm are financial processing or traffic monitoring. A distributed system is used to achieve the performance requisites. These same requisites force the distributed system not to store the events but to process them on the fly as they are received. These distributed systems are complex systems which require a considerably long time to learn and use. The majority of such distributed systems lack a declarative language in which to express the computation to perform over incoming events. In this work, a new SQL-like declarative language and a compiler have been developed. This compiler translates this new language to the distributed system native language. Due to its similarity with SQL a vast amount of developers who are already familiar with SQL will need little time to learn this language. Thus, this language reduces the execution failures at the time the programmer no longer needs to know every single detail of the underlying distributed system to submit a query.

DNA typing in thirty seconds with a microfabricated device

We report the development of a practical ultrafast allelic profiling assay for the analysis of short tandem repeats (STRs) by using a highly optimized microfluidic electrophoresis device. We have achieved baseline-resolved electrophoretic separations of single-locus STR samples in 30 sec. Analyses of PCR samples containing the four loci CSF1PO, TPOX, THO1, and vWA (abbreviated as CTTv) were performed in less than 2 min. This constitutes a 10- to 100-fold improvement in speed relative to capillary or slab gel systems. The separation device consists of a microfabricated channel 45 μm × 100 μm in cross section and 26 mm in length, filled with a replaceable polyacrylamide matrix operated under denaturing conditions at 50°C. A fluorescently labeled STR ladder was used as an internal standard for allele identification. Samples were prepared by standard procedures and only 4 μl was required for each analysis. The device is capable of repetitive operation and is suitable for automated high-speed and high-throughput applications.

Fluorescence energy transfer detection as a homogeneous DNA diagnostic method

A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.

Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors

We used plants as an in vivo pathogenesis model for the identification of virulence factors of the human opportunistic pathogen Pseudomonas aeruginosa. Nine of nine TnphoA mutant derivatives of P. aeruginosa strain UCBPP-PA14 that were identified in a plant leaf assay for less pathogenic mutants also exhibited significantly reduced pathogenicity in a burned mouse pathogenicity model, suggesting that P. aeruginosa utilizes common strategies to infect both hosts. Seven of these nine mutants contain TnphoA insertions in previously unknown genes. These results demonstrate that an alternative nonvertebrate host of a human bacterial pathogen can be used in an in vivo high throughput screen to identify novel bacterial virulence factors involved in mammalian pathogenesis.

Serial microanalysis of renal transcriptomes

Large-scale gene expression studies can now be routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues. In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced. To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1,000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution. It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods.

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.

Origin of nanomechanical cantilever motion generated from biomolecular interactions

Generation of nanomechanical cantilever motion from biomolecular interactions can have wide applications, ranging from high-throughput biomolecular detection to bioactuation. Although it has been suggested that such motion is caused by changes in surface stress of a cantilever beam, the origin of the surface-stress change has so far not been elucidated. By using DNA hybridization experiments, we show that the origin of motion lies in the interplay between changes in configurational entropy and intermolecular energetics induced by specific biomolecular interactions. By controlling entropy change during DNA hybridization, the direction of cantilever motion can be manipulated. These thermodynamic principles were also used to explain the origin of motion generated from protein–ligand binding.

Diversity Arrays: a solid state technology for sequence information independent genotyping

Here we present the successful application of the microarray technology platform to the analysis of DNA polymorphisms. Using the rice genome as a model, we demonstrate the potential of a high-throughput genome analysis method called Diversity Array Technology, DArT‘. In the format presented here the technology is assaying for the presence (or amount) of a specific DNA fragment in a representation derived from the total genomic DNA of an organism or a population of organisms. Two different approaches are presented: the first involves contrasting two representations on a single array while the second involves contrasting a representation with a reference DNA fragment common to all elements of the array. The Diversity Panels created using this method allow genetic fingerprinting of any organism or group of organisms belonging to the gene pool from which the panel was developed. Diversity Arrays enable rapid and economical application of a highly parallel, solid-state genotyping technology to any genome or complex genomic mixtures.

BodyMap incorporated PCR-based expression profiling data and a gene ranking system

BodyMap is a human and mouse gene expression database that is based on site-directed 3′-expressed sequence tags generated at Osaka University. To date, it contains more than 300 000 tag sequences from 64 human and 39 mouse tissues. For the recent release, the precise anatomical expression patterns for more than half of the human gene entries were generated by introduced amplified fragment length polymorphism (iAFLP), which is a PCR-based high-throughput expression profiling method. The iAFLP data incorporated into BodyMap describe the relative contents of more than 12 000 transcripts across 30 tissue RNAs. In addition, a newly developed gene ranking system helps users obtain lists of genes that have desired expression patterns according to their significance. BodyMap supports complete transfer of unique data sets and provides analysis that is accessible through the WWW at http://bodymap.ims.u-tokyo.ac.jp.

BIND—The Biomolecular Interaction Network Database

The Biomolecular Interaction Network Database (BIND; http://binddb.org) is a database designed to store full descriptions of interactions, molecular complexes and pathways. Development of the BIND 2.0 data model has led to the incorporation of virtually all components of molecular mechanisms including interactions between any two molecules composed of proteins, nucleic acids and small molecules. Chemical reactions, photochemical activation and conformational changes can also be described. Everything from small molecule biochemistry to signal transduction is abstracted in such a way that graph theory methods may be applied for data mining. The database can be used to study networks of interactions, to map pathways across taxonomic branches and to generate information for kinetic simulations. BIND anticipates the coming large influx of interaction information from high-throughput proteomics efforts including detailed information about post-translational modifications from mass spectrometry. Version 2.0 of the BIND data model is discussed as well as implementation, content and the open nature of the BIND project. The BIND data specification is available as ASN.1 and XML DTD.

The Medicago Genome Initiative: a model legume database

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume Medicago truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated Medicago functional genomics program at the Noble Foundation (http://www.noble .org), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

SpliceDB: database of canonical and non-canonical mammalian splice sites

A database (SpliceDB) of known mammalian splice site sequences has been developed. We extracted 43 337 splice pairs from mammalian divisions of the gene-centered Infogene database, including sites from incomplete or alternatively spliced genes. Known EST sequences supported 22 815 of them. After discarding sequences with putative errors and ambiguous location of splice junctions the verified dataset includes 22 489 entries. Of these, 98.71% contain canonical GT–AG junctions (22 199 entries) and 0.56% have non-canonical GC–AG splice site pairs. The remainder (0.73%) occurs in a lot of small groups (with a maximum size of 0.05%). We especially studied non-canonical splice sites, which comprise 3.73% of GenBank annotated splice pairs. EST alignments allowed us to verify only the exonic part of splice sites. To check the conservative dinucleotides we compared sequences of human non-canonical splice sites with sequences from the high throughput genome sequencing project (HTG). Out of 171 human non-canonical and EST-supported splice pairs, 156 (91.23%) had a clear match in the human HTG. They can be classified after sequence analysis as: 79 GC–AG pairs (of which one was an error that corrected to GC–AG), 61 errors corrected to GT–AG canonical pairs, six AT–AC pairs (of which two were errors corrected to AT–AC), one case was produced from a non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two other cases left of supported non-canonical splice pairs. The information about verified splice site sequences for canonical and non-canonical sites is presented in SpliceDB with the supporting evidence. We also built weight matrices for the major splice groups, which can be incorporated into gene prediction programs. SpliceDB is available at the computational genomic Web server of the Sanger Centre: http://genomic.sanger.ac.uk/spldb/SpliceDB.html and at http://www.softberry.com/spldb/SpliceDB.html.

trEST, trGEN and Hits: access to databases of predicted protein sequences

High throughput genome (HTG) and expressed sequence tag (EST) sequences are currently the most abundant nucleotide sequence classes in the public database. The large volume, high degree of fragmentation and lack of gene structure annotations prevent efficient and effective searches of HTG and EST data for protein sequence homologies by standard search methods. Here, we briefly describe three newly developed resources that should make discovery of interesting genes in these sequence classes easier in the future, especially to biologists not having access to a powerful local bioinformatics environment. trEST and trGEN are regularly regenerated databases of hypothetical protein sequences predicted from EST and HTG sequences, respectively. Hits is a web-based data retrieval and analysis system providing access to precomputed matches between protein sequences (including sequences from trEST and trGEN) and patterns and profiles from Prosite and Pfam. The three resources can be accessed via the Hits home page (http://hits.isb-sib.ch).

Alkaline-mediated differential interaction (AMDI): A simple automatable single-nucleotide polymorphism assay

The key requirements for high-throughput single-nucleotide polymorphism (SNP) typing of DNA samples in large-scale disease case-control studies are automatability, simplicity, and robustness, coupled with minimal cost. In this paper we describe a fluorescence technique for the detection of SNPs that have been amplified by using the amplification refractory mutation system (ARMS)-PCR procedure. Its performance was evaluated using 32 sequence-specific primer mixes to assign the HLA-DRB alleles to 80 lymphoblastoid cell line DNAs chosen from our database for their diversity. All had been typed previously by alternative methods, either direct sequencing or gel electrophoresis. We believe the detection system that we call AMDI (alkaline-mediated differential interaction) satisfies the above criteria and is suitable for general high-throughput SNP typing.