RESUMEN
Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In humans, four AP isozymes have been identified-one tissue-nonspecific (TNAP) and three tissue-specific-named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endotoxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with specificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.
Asunto(s)
Acetanilidas/química , Acetanilidas/farmacología , Fosfatasa Alcalina/antagonistas & inhibidores , Sulfonamidas/química , Sulfonamidas/farmacología , Acetanilidas/aislamiento & purificación , Animales , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Isoformas de Proteínas/química , Sulfonamidas/aislamiento & purificaciónRESUMEN
Microfluidic-based devices have allowed miniaturization and increased parallelism of many common functions in biological assays; however, development of a practical technology for microfluidic-based fluorescence-activated cell sorting has proved challenging. Although a variety of different physical on-chip switch mechanisms have been proposed, none has satisfied simultaneously the requirements of high throughput, purity, and recovery of live, unstressed mammalian cells. Here we show that optical forces can be used for the rapid (2-4 ms), active control of cell routing on a microfluidic chip. Optical switch controls reduce the complexity of the chip and simplify connectivity. Using all-optical switching, we have implemented a fluorescence-activated microfluidic cell sorter and evaluated its performance on live, stably transfected HeLa cells expressing a fused histone-green fluorescent protein. Recovered populations were verified to be both viable and unstressed by evaluation of the transcriptional expression of two genes, HSPA6 and FOS, known indicators of cellular stress.
Asunto(s)
Separación Celular/métodos , Citometría de Flujo/métodos , Microfluídica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de la Célula , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Histonas/química , Humanos , Rayos Láser , Polipropilenos/química , ARN Mensajero/metabolismo , Semiconductores , Espectrometría de Fluorescencia/métodos , Temperatura , Transcripción Genética , TransfecciónRESUMEN
Microelectronic arrays have been developed for DNA hybridization analysis of point mutations, single nucleotide polymorphisms, short tandem repeats and gene expression. In addition to a variety of molecular biology and genomic research applications, such devices will also be used for infectious disease detection, genetic and cancer diagnostics, and pharmacogenomic applications. These microelectronic array devices are able to produce defined electric fields on their surfaces that allow charged molecules and other entities to be transported to or from any test site or micro-location on the planar surface of the device. These molecules and entities include DNA, RNA, proteins, enzymes, antibodies and cells. Electronic-based molecule addressing and hybridization can then be carried out, where the electric field is now used to greatly accelerate the hybridization reactions that occur on the selected test sites. When reversed, the electric field can be used to provide an additional parameter for improved hybridization. Special low-conductance buffers have been developed that provide for the rapid transport of the DNA molecules and facilitate the electronic hybridization reactions under conditions that do not support hybridization. Important to the device function is the permeation layer that overcoats the underlying microelectrodes. Generally composed of a porous hydrogel material impregnated with attachment chemistry, this permeation layer prevents the destruction of analytes at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization and affinity reactions, and serves as a support structure for attaching DNA probes and other molecules to the array. The microelectronic chip or array device is incorporated into a cartridge package (NanoChip trade mark cartridge) that provides the electronic, optical, and fluidic interfacing. A complete instrument system (NanoChip trade mark Molecular Biology Workstation) provides a chip loader, fluorescent reader, computer control interface and data display screen. The probe loader component allows DNA probes or target molecules (polymerase chain reactions amplicons, genomic DNA, RNA, etc.) to be selectively addressed to the array test sites, providing the end-user with 'make your own chip' capabilities. The electronic hybridization can then be carried out and the chip analyzed using a fluorescent detector system. In addition to carrying out rapid, accurate and highly reliable genotyping (point mutations, single nucleotide polymorphisms, short tandem repeats), other future applications include gene expression analysis, or on-chip amplification, immunoassays and cell separation and selection. Smaller and more compact systems are also being designed for portable sample to answer and point of care diagnostics.
Asunto(s)
Genotipo , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Mutación Puntual , Polimorfismo Genético , Sondas de ADN , Electrónica , Factor V/genética , Hemocromatosis/genética , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
An integrated, stacked microlaboratory for performing automated electric-field-driven immunoassays and DNA hybridization assays was developed. The stacked microlaboratory was fabricated by orderly laminating several different functional layers (all 76 x 76 mm(2)) including a patterned polyimide layer with a flip-chip bonded CMOS chip, a pressure sensitive acrylic adhesive (PSA) layer with a fluidic cutout, an optically transparent polymethyl methacrylate (PMMA) film, a PSA layer with a via, a patterned polyimide layer with a flip-chip bonded silicon chip, a PSA layer with a fluidic cutout, and a glass cover plate layer. Versatility of the stacked microlaboratory was demonstrated by various automated assays. Escherichia coli bacteria and Alexa-labeled protein toxin staphylococcal enterotoxin B (SEB) were detected by electric-field-driven immunoassays on a single chip with a specific-to-nonspecific signal ratios of 4.2:1 and 3.0:1, respectively. Furthermore, by integrating the microlaboratory with a module for strand displacement amplification (SDA), the identification of the Shiga-like toxin gene (SLT1) from E. coli was accomplished within 2.5 h starting from a dielectrophoretic concentration of intact E. coli bacteria and finishing with an electric-field-driven DNA hybridization assay, detected by fluorescently labeled DNA reporter probes. The integrated microlaboratory can be potentially used in a wide range of applications including detection of bacteria and biowarfare agents, and genetic identification.
Asunto(s)
Sondas de ADN/química , ADN Bacteriano/análisis , Enterotoxinas/análisis , Inmunoensayo/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Toxina Shiga I/análisis , Guerra Biológica/prevención & control , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , ADN Bacteriano/genética , Electroquímica/métodos , Campos Electromagnéticos , Electroforesis/métodos , Diseño de Equipo , Escherichia coli/clasificación , Escherichia coli/genética , Inmunoensayo/métodos , Miniaturización , Técnicas de Amplificación de Ácido Nucleico/métodos , Hibridación de Ácido Nucleico , Semiconductores , Toxina Shiga I/genéticaRESUMEN
To facilitate quantitation of cellular apoptotic responses to various antineoplastic agents, a laser-based technology, Optophoresis, has been developed to provide analysis of cells without any need for labeling or cell processing. Optophoresis is defined as the analysis of the motion of cells, where the motion is either induced or modified by a moving optical gradient field, which produces radiation pressure forces on the cells in an aqueous suspension. Quantitation of the induced motion provides a basis for distinguishing one population of cells from another. One Optophoretic technique, Fast Scan, measures the distribution of distances traversed by a population of cells when exposed to a fast-moving optical gradient. Fast Scan was validated using a cell-based model of chronic myeloid leukemia treated with Gleevec, a specific inhibitor of aberrant Bcr-Abl protein kinase. The Optophoretic measurements were quantitatively comparable to reference assays with regard to drug selectivity and potency and to target specificity, demonstrating the suitability of this technology for pharmaceutical and clinical applications.