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Metallic nanoparticles suspended in aqueous solutions and functionalized with chemical and biological surface coatings are important elements in basic and applied nanoscience research. Many applications require an understanding of the electrokinetic or colloidal properties of such particles. We describe the results of experiments to measure the zeta potential of metallic nanorod particles in aqueous saline solutions, including the effects of pH, ionic strength, metallic composition, and surface functionalization state. Particle substrates tested include gold, silver, and palladium monometallic particles as well as gold/silver bimetallic particles. Surface functionalization conditions included 11-mercaptoundecanoic acid (MUA), mercaptoethanol (ME), and mercaptoethanesulfonic acid (MESA) self-assembled monolayers (SAMs), as well as MUA layers subsequently derivatized with proteins. For comparison, we present zeta potential data for typical charge-stabilized polystyrene particles. We compare experimental zeta potential data with theoretically predicted values for SAM-coated and bimetallic particles. The results of these studies are useful in predicting and controlling the aggregation, adhesion, and transport of functionalized metallic nanoparticles within microfluidic devices and other systems.
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Electroforesis/métodos , Nanopartículas del Metal/química , Electricidad , Ácidos Grasos/química , Oro/química , Modelos Teóricos , Compuestos de Sulfhidrilo/químicaRESUMEN
Surface-enhanced Raman scattering (SERS) is an optical detection technique that offers advantages over traditional assay detection technologies, such as fluorescence and chemiluminescence. These advantages include sensitivity, high levels of multiplexing, robustness and ability to perform detection in blood and other biological matrices. Here, we report on the growing field of SERS-active nanoparticles as a novel method for detection, with special emphasis on their use in the field of oncology. We discuss examples of SERS-active nanoparticles used in an assay for PSA, BRCA1 and Her-2, along with examples of nucleic-acid detection. We present data on a novel homogeneous, single-tube, rapid assay for nucleic acid detection and show how it will benefit the oncology community.
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Aumento de la Imagen/métodos , Nanomedicina/tendencias , Nanopartículas , Neoplasias/diagnóstico , Resonancia por Plasmón de Superficie/métodos , Medios de Contraste , HumanosRESUMEN
We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable no-wash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase DeltaG of folding in 500 mM buffered NaCl of approximately -4 kcal/mol performed better than those with DeltaG > -2 or < -6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 degrees C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with Kd = 1.7 +/- 0.3 nM. The detection limit was approximately 100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.
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ADN Viral/análisis , VIH/química , Hepacivirus/química , Metales/química , Nanoestructuras/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Oro/química , Humanos , Técnicas de Sonda Molecular/instrumentación , Sondas Moleculares/química , Concentración Osmolar , Sensibilidad y Especificidad , Plata/química , Cloruro de Sodio/química , Propiedades de Superficie , TermodinámicaRESUMEN
Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.
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ADN/análisis , Nanopartículas/química , Nanotecnología/métodos , Polimorfismo de Nucleótido Simple/genética , ADN/genética , Genotipo , Humanos , Nanotubos/química , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y EspecificidadRESUMEN
We have developed striped metal nanoparticles, Nanobarcodes particles, which can act as encoded substrates in multiplexed assays. These particles are metallic, encodeable, machine-readable, durable, submicron-sized tags. The power of this technology is that the particles are intrinsically encoded by virtue of the difference in reflectivity of adjacent metal stripes. This chapter describes protocols for the attachment of biological molecules, and the subsequent use of the Nanobarcodes particles in bioassays.
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Perfilación de la Expresión Génica/métodos , Nanoestructuras/química , Nanotecnología/métodos , Oligonucleótidos/química , Animales , HumanosRESUMEN
In this paper we describe a molecular beacon format assay in which encoded nanowire particles are used to achieve multiplexing. We demonstrate this principle with the detection of five viral pathogens; Hepatitis A virus, Hepatitis C virus, West Nile Virus, Human Immune Deficiency virus and Severe Acute Respiratory Syndrome virus. Oligonucleotides are designed complementary to a target sequence of interest containing a 3' universal fluorescence dye. A 5' thiol causes the oligonucleotides to self-assemble onto the metal nanowire. The single-stranded oligonucleotide contains a self-complementary hairpin stem sequence of 10 bases that forces the 3' fluorophore to come into contact with the metallic nanowire surface, thereby quenching the fluorescence. Upon addition of target DNA, there is hybridization with the complementary oligonucleotides. The resulting DNA hybrid is rigid, unfolds the hairpin structure, and causes the fluorophore to be moved away from the surface such that it is no longer quenched. By using differently encoded nanowires, each conjugated with a different oligonucleotide sequence, multiplexed DNA assays are possible using a single fluorophore, from a multiplexed RT-PCR reaction.
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Genes involved in the testosterone biosynthetic pathway - such as CYP17A1, CYP3A4, and SRD5A2 - represent strong candidates for affecting prostate cancer. Previous work has detected associations between individual variants in these three genes and prostate cancer risk and aggressiveness. To more comprehensively evaluate CYP17A1, CYP3A4, and SRD5A2, we undertook a two-phase study of the relationship between their genotypes/haplotypes and prostate cancer. Phase I of the study first searched for single-nucleotide polymorphisms (SNPs) in these genes by resequencing 24 individuals from the Coriell Polymorphism Discovery Resource, 92-110 men from prostate cancer case-control sibships, and by leveraging public databases. In all, 87 SNPs were discovered and genotyped in 276 men from case-control sibships. Those SNPs exhibiting preliminary case-control allele frequency differences, or distinguishing (ie, 'tagging') common haplotypes across the genes, were identified for further study (24 SNPs in total). In Phase II of the study, the 24 SNPs were genotyped in an additional 841 men from case-control sibships. Finally, associations between genotypes/haplotypes in CYP17A1, CYP3A4, and SRD5A2 and prostate cancer were evaluated in the total case-control sample of 1117 brothers from 506 sibships. Family-based analyses detected associations between prostate cancer risk or aggressiveness and a number of CYP3A4 SNPs (P-values between 0.006 and 0.05), a CYP3A4 haplotype (P-values 0.05 and 0.009 in nonstratified and stratified analysis, respectively), and two SRD5A2 SNPs in strong linkage disequilibrium (P=0.02). Undertaking a two-phase study comprising SNP discovery, haplotype tagging, and association analyses allowed us to more fully decipher the relation between CYP17A1, CYP3A4, and SRD5A2 and prostate cancer.
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3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Sistema Enzimático del Citocromo P-450/genética , Haplotipos , Neoplasias de la Próstata/genética , Esteroide 17-alfa-Hidroxilasa/genética , Estudios de Casos y Controles , Citocromo P-450 CYP3A , Genotipo , Humanos , Masculino , Polimorfismo de Nucleótido SimpleRESUMEN
A key challenge in measuring gene expression changes in the lung in response to site-selective toxicants is differentiating between target and nontarget areas. The toxicity for the cytotoxicant 1-nitronaphthalene is highly localized in the airway epithelium. Target cells comprise but a fraction of the total lung cell mass; measurements from whole lung homogenates are not likely to reflect what occurs at the target site. Additionally, the use of generic microarrays to measure expression in airway epithelium may not provide a good representation of transcripts present at the site of toxic action. cDNA libraries from airway and alveolar subcompartments of rat lung were sequenced for the development of a custom microarray representative of these lung regions. We identified 7,460 nonredundant rat lung sequences. Nearly 30% of the sequences on this array are not present on the Affymetrix Rat GeneChip 230. A 20,000-element microarray was developed that delineates differences in gene expression between subcompartments. This is the first in a series of articles employing this microarray for detecting gene expression changes during acute injury produced by 1-nitronaphthalene and subsequent repair.
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Carcinógenos/toxicidad , Perfilación de la Expresión Génica , Expresión Génica/efectos de los fármacos , Pulmón/efectos de los fármacos , Naftalenos/toxicidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Células Epiteliales/metabolismo , Biblioteca de Genes , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratas , Ratas Sprague-Dawley , Análisis de Secuencia de ADNRESUMEN
This review covers the emerging field of nanobiotechnology, in which nanoparticles are applied to the analysis of biomolecules. Nanoparticles can be used in a variety of bioanalytical formats, and this review discusses four classes of use. First, nanoparticles as quantitation tags, such as the optical detection of quantum dots and the electrochemical detection of metallic nanoparticles. Second, encoded nanoparticles as substrates for multiplexed bioassays, such as striped metallic nanoparticles. Third, nanoparticles that leverage signal transduction, for example in colloidal gold-based aggregation assays. Fourth, functional nanoparticles that exploit specific physical or chemical properties of nanoparticles to carry out novel functions, such as the catalysis of a biological reaction. In addition, the review discusses the next generation of nanoparticles that will be utilized in the life sciences, such as nanodots and carbon nanotubes.
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Técnicas Biosensibles , Nanotecnología/métodos , Biotecnología/métodos , Puntos Cuánticos , Transducción de SeñalAsunto(s)
Teorema de Bayes , Análisis de Secuencia por Matrices de Oligonucleótidos , Toxicología/métodos , Animales , Etiquetas de Secuencia Expresada , Regulación de la Expresión Génica/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador , Hígado/efectos de los fármacos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Toxinas Biológicas/metabolismo , Toxinas Biológicas/farmacologíaRESUMEN
MOTIVATION: In many microarray experiments, relatively few intra- and inter-array replicate measurements are made due to significant cost limitations and sample availability. Compounding this problem is a lack of robust statistical methods for analyzing gene expression data with limited experimental replicates. As a result, the interpretation of the results of these experiments are difficult with little understanding of the probability of type I and type II errors. RESULTS: The variability in a series of replicate microarray measurements was modelled using a combination of parametric and non-parametric methods. A 3-dimensional surface was created for the conditional distribution of the variability given the mean signal intensity in both the Cy3 and Cy5 channels. The results were used as the basis for developing statistical methods for analyzing gene expression data with limited experimental replicates. AVAILABILITY: The statistical analysis scripts are available upon request.
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ADN/genética , Expresión Génica , Modelos Genéticos , Modelos Estadísticos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Simulación por Computador , Replicación del ADN/genética , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/estadística & datos numéricos , Regulación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The Ahr locus encodes for the aryl hydrocarbon receptor (AHR), which plays an important toxicological and developmental role. Sequence variation in this gene was studied in 13 different mouse lines that included eight laboratory strains, two Mus musculus subspecies and three additional Mus species. The data presented represent the largest study of sequence variation across multiple mouse lines in a single gene (approximately equal to 15.9 kb/mouse line). Among all mice, the average frequency of all polymorphisms in the intronic regions was 20.3 variants/kb and the average exonic frequency was 14.1 variants/kb. For substitutions alone, the average frequencies in the intronic and exonic regions for all mice were 13.3 and 8.9 substitutions/kb, respectively. Between laboratory strains, the average intronic and exonic frequencies for all polymorphisms dropped to 5.4 and 2.9 variants/kb, respectively. There were 111 non-synonymous polymorphisms that resulted in 42 different amino acid changes, of which only 10 amino acid changes had been previously identified. Based on the nucleotide sequence, the phylogenetic history of the gene showed mice from the Ahr(b2) and Ahr(d) alleles in separate branches while mice from the Ahr(b1) and Ahr(b3) alleles exhibited a more complex history. Evolutionarily, the AHR protein as a whole appears to be under purifying selective pressure (K(a) : K(s) ratio = 0.237). Despite significant functional constraint in the basic helix-loop-helix and PAS domains, ligand binding is not constrained to the high-affinity allele, which supports further the role of the AHR in development and its importance beyond the adaptive response to environmental toxicants.
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Variación Genética , Ratones Endogámicos/genética , Polimorfismo Genético , Receptores de Hidrocarburo de Aril/genética , Secuencia de Aminoácidos , Animales , Evolución Molecular , Ligamiento Genético , Ratones , Datos de Secuencia Molecular , Filogenia , Selección Genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Traditional models of toxicity have relied on dissecting chemical action into pharmacokinetic and pharmacodynamic processes. However, the integration of genomic information with toxicology will enhance our basic understanding of these processes and significantly change the way we apply toxicological information to risk assessment and regulatory problems. In this article, we summarize the application of gene expression information and polymorphism discovery to four areas in toxicology: toxicity testing, cross-species extrapolation, understanding mechanism of action, and susceptibility.