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1.
Anal Chem ; 95(32): 11868-11873, 2023 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-37535807

RESUMO

Protein glycosylation is a crucial factor that must be evaluated in biological pharmaceuticals. The glycoform profile of a protein can vary depending on the conditions of the cultivation, purification process, and the selection of a host cell. Lectin microarrays are reliable bioanalytical methods used in the early phases of bioprocesses for the detection of glycosylation. The concept of a fully automated glycan detection with a bead array has been previously reported; however, no simple system has been constructed on fluorescence-based detection using a microarray. Here, we present a fully automated detection system equipped with a novel fluorescence detector for a 13-lectin bead array with a single tip. The lattice-like arrangement of a set of fibers proximate to the tip of the light emitting diode and photomultiplier tube detector minimized the noise caused by the reflection of incident light on the plastic capillary tip and bead. A unique rolling-circle fiber unit with quadruple lattices stacked in two layers realizes the 8-parallel automeasurement with a drastic reduction in scanning time and machine size. The 8-glycan profiles obtained automatically within 25 min were identical with those obtained with the conventional lectin microarray after overnight incubation. The signals obtained were represented as lectin dotcodes. Therefore, autolectin dotcoding assisted by the twin 8 legs named as "detection and irradiation octopuses" may be a rapid glyco-evaluation system during the production and development of biopharmaceuticals.

2.
Biomedicines ; 10(9)2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36140390

RESUMO

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. METHODS: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. RESULTS: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. CONCLUSIONS: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories.

3.
Sci Rep ; 10(1): 18718, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127950

RESUMO

Extracellular vesicles (EVs), such as exosomes, have garnered increasing interest because of their potential clinical applications that range from diagnostics to therapeutics. The development of an automated and reproducible EV purification platform would therefore aid the introduction of EV biomarkers and therapies into the clinic. Here, we demonstrate that K8- as well as K-16 peptides (containing 8 and 16 lysine residues with dissociation constants of 102 nM and 11.6 nM for phosphatidylserine, respectively) immobilized on magnetic beads can capture small EVs (< 0.2 µm) from culture supernatants of MCF7 human breast cancer cells. Importantly, the bound EVs could be dissociated from the beads under mild conditions (e.g. 0.5 M NaCl), and the isolated EVs had the typical shapes of EVs under SEM and TEM with a mean particle size of 99 nm. Using the peptide-immobilized beads, we adapted a pre-existing bench top instrument for magnetic separation to perform automated EV purification with higher purity and yield than that obtained using the standard ultracentrifugation method.


Assuntos
Vesículas Extracelulares/química , Peptídeos/química , Fosfolipídeos/química , Biomarcadores/química , Exossomos/química , Humanos , Separação Imunomagnética , Lipídeos/química , Lisina/química , Células MCF-7 , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Fosfatidilserinas/química , Reprodutibilidade dos Testes
4.
Anal Chem ; 91(17): 11162-11169, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31405276

RESUMO

A quantitative description of glyco-alteration/differences in diseases can lead to the development of a diagnostic agent for use in vitro to monitor the degree of change in target glycoproteins. Analytical systems have been developed along with the progress of omics-oriented technologies. For clinical implementation, their full automation is required with an apparatus that is simple to operate. Here, we report an automatic analysis system for quantitative characterization of glyco-alteration/differences that depends on the unique strategy of "bead arrays in a single tip." The alternative lectin array can obtain a minimum characterization of the glycan profile for nanogram quantities of an endogenous glycoprotein. A simple autopipetting robot produces the precise chemiluminescence detection of glycan-lectin interactions with a wide dynamic range that is superior to fluorescence-based lectin arrays. The tip-based array format enables automatic glycan profiling from sample pretreatment to detection with low variation and linear detection, which may facilitate the use of this lectin array in clinical practice.


Assuntos
Automação , Glicoproteínas/análise , Lectinas/química , Fluorescência , Espectrometria de Massas , Análise em Microsséries
5.
J Biosci Bioeng ; 110(4): 505-8, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20547337

RESUMO

A low-cost and simple on-site technique for genotyping single nucleotide polymorphisms (SNPs) was developed. The technique is based on allele-specific primer PCR and the recently developed bead arrays in a single tip technique. The performance of the method was verified by genotyping four SNPs that correlate with cardiovascular diseases.


Assuntos
Automação , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Primers do DNA
6.
Transl Res ; 153(2): 91-6, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19138654

RESUMO

In this study, we aimed to explore whether interleukin-18 (IL-18) gene-promoter polymorphisms are associated with the outcome of hepatitis B virus (HBV) infection. In all, 204 chronically HBV-infected patients were recruited in this study. Of the 204 HBV-infected patients, 43 were considered to be inactive HBV carriers based on the sustained normalization of serum alanine aminotransferase (ALT) together with seropositivity for the antibody to hepatitis B e-antigen (anti-HBe). A total of 161 patients were found to have chronic progressive liver disease, which included cirrhosis. In these HBV-infected patients, the frequencies of AA genotype of IL-18 gene-promoter polymorphisms at position -607 and C allele at position -137 were significantly higher in inactive HBV carriers compared with those in patients with chronic progressive liver disease. These polymorphisms of the IL-18 promoter regions (-607 and -137) could be associated with different outcomes of HBV infection.


Assuntos
Hepatite B/genética , Interleucina-18/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Portador Sadio , Progressão da Doença , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética
7.
Appl Environ Microbiol ; 74(16): 5139-45, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18567691

RESUMO

Biotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) or biotin carboxyl carrier protein (BCCP) on the surface of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. BAP-displaying BacMPs (BAP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. Then the in vitro biotinylation of BAP-BacMPs was confirmed using alkaline phosphatase-labeled antibiotin antibody. In contrast, BacMPs displaying the intact 149 residues of AMB-1 BCCP (BCCP-BacMPs) and displaying the COOH-terminal 78 residues of BCCP (BCCP78-BacMPs) were biotinylated in AMB-1 cells. The in vivo biotinylation of BCCP-BacMPs and BCCP78-BacMPs was thought to be performed by endogenous AMB-1 biotin ligase. Streptavidin was introduced onto biotinylated BacMPs by simple mixing. In an analysis using tetramethyl rhodamine isocyanate-labeled streptavidin, approximately 15 streptavidin molecules were shown to be immobilized on a single BCCP-BacMP. Furthermore, gold nanoparticle-BacMP composites were constructed via the biotin-streptavidin interaction. The conjugation system developed in this work provides a simple, low-cost method for producing biotin- or streptavidin-labeled magnetic nanoparticles. Various functional materials can be site selectively immobilized on these specially designed BacMPs. By combining the site-selective biotinylation technology and the protein display technology, more innovative and attractive magnetic nanomaterials can be constructed.


Assuntos
Biotinilação/métodos , Magnetismo , Magnetospirillum/química , Nanopartículas/química , Estreptavidina/química , Acetil-CoA Carboxilase/química , Carbono-Nitrogênio Ligases/química , Proteínas de Transporte/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Ácido Graxo Sintase Tipo II , Proteínas Repressoras/química
8.
Biotechnol Appl Biochem ; 51(Pt 2): 111-7, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18248326

RESUMO

Previously we developed MagSNiPer, an SNP (single nucleotide polymorphism) genotyping method. In the present paper we show development of an automated system for MagSNiPer, namely MagSNiPer Station, and its application for quantitative discrimination of Dehalococcoides species, which perform anaerobic dechlorination of chloroethenes. MagSNiPer Station is equipped with a thermal cycler, a tip stand, a microtitre-plate automated stacker, an eight-channel tip dispenser, a magnetic separation unit for Magtration technology, and a chemiluminescence detector. It can automatically perform all processes required for SNP genotyping by MagSNiPer. A primer was designed for discriminating single nucleotide difference between 16 S rRNA genes of Dehalococcoides ethenogenes and Dehalococcoides BAV1. Chemiluminescence intensities for the 16 S rRNA genes obtained by MagSNiPer were proportional to their quantity. MagSNiPer analysis of 16 S rRNA genes amplified on the DNA purified from groundwater gave a ratio of these two 16 S rRNA genes similar to that obtained by cloning and sequencing. MagSNiPer is much easier, more rapid and more cost-effective than conventional sequencing. Compared with denaturing gradient-gel electrophoresis, MagSNiPer has the advantage of being quantitative. Therefore, by applying MagSNiPer at several sites where single base differences exist among Dehalococcoides species, it is possible to analyse Dehalococcoides consortia with ease, yielding useful information on anaerobic bioremediation of chloroethenes.


Assuntos
Chloroflexi/genética , Polimorfismo de Nucleotídeo Único/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/instrumentação , Análise de Sequência de DNA/métodos , Genótipo , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Especificidade da Espécie
9.
Anal Chim Acta ; 597(2): 331-9, 2007 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-17683747

RESUMO

Magnetic beads have served as a conventional bioassay platform in biotechnology. In this study, a fully automated immunoassay was performed using novel nano- and microbead-composites constructed by assembling nano-magnetic beads onto polystyrene microbeads, designated 'Beads on Beads'. Nano-sized bacterial magnetic particles (BacMPs) displaying the immunoglobulin G (IgG)-binding domain of protein A (ZZ domain) were used for the construction of 'Beads on Beads' via the interaction of biotin-streptavidin. The efficient assembly of 'Beads on Beads' was performed by gradual addition of biotin-labeled BacMPs onto streptavidin-coated polystyrene microbeads. Approximately 2000 BacMPs were uniformly assembled on a single microbead without aggregation. The constructed 'Beads on Beads' were magnetized and separated from the suspension by using an automated magnetic separation system with a higher efficiency than BacMPs alone. Furthermore, fully automated detection of prostate-specific antigens was performed with the detection limit of 1.48 ng mL(-1). From this preliminary assay, it can be seen that 'Beads on Beads' could be a powerful tool in the development of high-throughput, fully automated multiplexed bioassays.


Assuntos
Imunoensaio/instrumentação , Imunoensaio/métodos , Magnetismo , Nanopartículas/ultraestrutura , Poliestirenos , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/imunologia , Anticorpos/imunologia , Humanos , Microscopia Eletrônica de Varredura
10.
Anal Biochem ; 367(1): 79-86, 2007 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-17570330

RESUMO

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Polimorfismo de Nucleotídeo Único , Receptores Opioides mu/genética , Alelos , Sequência de Bases , DNA/genética , DNA/isolamento & purificação , Desenho de Equipamento , Genótipo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de Oligonucleotídeos/genética
11.
Biotechnol Bioeng ; 98(2): 420-8, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17335059

RESUMO

Biological and medical importance of the single nucleotide polymorphism (SNP) has led to development of a wide variety of methods for SNP typing. Aiming for establishing highly reliable and fully automated SNP typing, we have developed the adapter ligation method in combination with the paramagnetic beads handling technology, Magtration(R). The method utilizes sequence specific ligation between the fluorescently labeled adapter and the sample DNAs at the cohesive end produced by a type IIS restriction enzyme. Evaluation of the method using human genomic DNA showed clear discrimination of the three genotypes without ambiguity using the same reaction condition for any SNPs examined. The operations following PCR amplification were automatically performed by the Magtration(R)-based robot that we have previously developed. Multiplex typing of two SNPs in a single reaction by using four fluorescent dyes was successfully preformed at the almost same sensitivity and reliability as the single typing. These results demonstrate that the automated paramagnetic beads handling technology, Magtration(R), is highly adaptable to the automated SNP analysis and that our method best fits to an automated in-house SNP typing for laboratory and medical uses.


Assuntos
Genótipo , Magnetismo , Reação em Cadeia da Polimerase/instrumentação , Polimorfismo de Nucleotídeo Único/genética , Robótica/instrumentação , Análise de Sequência de DNA/instrumentação , Desenho de Equipamento/instrumentação , Testes Genéticos , Genoma Humano , Humanos
12.
J Biosci Bioeng ; 102(5): 474-7, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17189179

RESUMO

A single-nucleotide-polymorphism-typing method using a novel three-dimensional DNA microarray, Bio-Strand, is promising because it is rapid, inexpensive and easily automated. It has been developed with the intent to overcome the drawbacks of conventional DNA microarrays, which use flat surfaces and impermeable materials such as glass slides; Bio-Strand as a novel DNA microarray, with its permeability, has a significantly improved stability compared with conventional DNA microarrays that use impermeable materials. In this study, we have developed a simple method of pretreating a polyamide monofilament to increase its surface area and to make it permeable, which makes Bio-Strand more sensitive and stable, allowing it to be adapted for clinical diagnostic applications. The fluorescence signal obtained with a nylon 6 monofilament pretreated under optimal conditions (hydrolysis by 5 M HCl/ethanol followed by washing with 50% ethanol and 100% ethanol) was significantly stronger than that obtained with an untreated monofilament.


Assuntos
Ácido Clorídrico/farmacologia , Nylons/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
13.
J Biosci Bioeng ; 99(2): 120-4, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16233767

RESUMO

Previously, we developed a novel three-dimensional microarray system called Bio-Strand, which may be used in various applications including single nucleotide polymorphisms genotyping. In Bio-Strand, samples for detection are immobilized on a one-dimensional thread, which is wound around a cylinder-shaped core to form a three-dimensional thread-and-core structure. The thread-and-core structure is then inserted into a plastic pipette tip, where hybridization and detection are performed. In this study, we have developed an automation system, NIAGALA Bio-Station SDx, which enables automated hybridization and detection during the genotyping procedure using Bio-Strand. Using this system, we have performed the single nucleotide polymorphism (SNP) genotyping of CYP2C, one of the important human cytochrome P450 genes and the results were completely consistent with the genotyping results determined by the TaqMan method.


Assuntos
Algoritmos , Sistema Enzimático do Citocromo P-450/genética , Análise Mutacional de DNA/métodos , Hibridização in Situ Fluorescente/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Polimorfismo de Nucleotídeo Único/genética , Manejo de Espécimes/instrumentação , Inteligência Artificial , Desenho de Equipamento , Análise de Falha de Equipamento , Hibridização in Situ Fluorescente/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reconhecimento Automatizado de Padrão/métodos , Robótica/métodos , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos
14.
Biosci Biotechnol Biochem ; 69(8): 1616-9, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16116296

RESUMO

The lectin-antibody enzyme immunoassay of the alphafetoprotein-L3 carbohydrate chain, a tumor marker of liver cancer, has not been automated. We improved the technique of the assay for automation. Consequently, alphafetoprotein-L3 and total alphafetoprotein were detected with two lectins using an automatic paramagnetic bead handling robot. This indicates that the improved method is potentially applicable to the automated enzyme immunoassay robot.


Assuntos
Carboidratos/análise , Técnicas Imunoenzimáticas/instrumentação , Robótica , alfa-Fetoproteínas/análise , alfa-Fetoproteínas/química , alfa-Fetoproteínas/imunologia
15.
Anal Biochem ; 341(1): 77-82, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15866530

RESUMO

We have developed a new method for typing single nucleotide polymorphisms (SNPs), MagSNiPer, based on single base extension, magnetic separation, and chemiluminescence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3' terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemiluminescence. The incorporation of labeled ddNTP is monitored by chemiluminescence induced by alkaline phosphatase. MagSNiPer is a simple and robust SNP typing method with a wide dynamic range and high sensitivity. Using MagSNiPer, we could perform SNP typing with as little as 10(-17) mol of template DNA.


Assuntos
Medições Luminescentes , Polimorfismo de Nucleotídeo Único , Adulto , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP2D6/genética , Marcadores Genéticos , Humanos , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos
16.
J Biol Chem ; 280(10): 9698-705, 2005 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-15598657

RESUMO

L-rhamnose is an essential component of the cell wall and plays roles in mediating virulence and adhesion to host tissues in many microorganisms. Glucose-1-phosphate thymidylyltransferase (RmlA, EC 2.7.7.24) catalyzes the first reaction of the four-step pathway of L-rhamnose biosynthesis, producing dTDP-D-glucose from dTTP and glucose-1-phosphate. Three RmlA homologues of varying size have been identified in the genome of a thermophilic archaeon, Sulfolobus tokodaii strain 7. In this study, we report the heterologous expression of the largest homologue (a 401 residue-long ST0452 protein) and characterization of its thermostable activity. RmlA enzymatic activity of this protein was detected from 65 to 100 degrees C, with a half-life of 60 min at 95 degrees C and 180 min at 80 degrees C. Analysis of a deletion mutant lacking the 170-residue C-terminal domain indicated that this region has an important role in the thermostability and activity of the protein. Analyses of substrate specificity indicated that the enzymatic activity of the full-length protein is capable of utilizing alpha-D-glucose-1-phosphate and N-acetyl-D-glucosamine-1-phosphate but not alpha-D-glucosamine-1-phosphate. However, the protein is capable of utilizing all four deoxyribonucleoside triphosphates and UTP. Thus, the ST0452 protein is an enzyme containing both glucose-1-phosphate thymidylyltransferase and N-acetyl-D-glucosamine-1-phosphate uridylyltransferase activities. This is the first report of a thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities.


Assuntos
Nucleotidiltransferases/metabolismo , Sulfolobus/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Primers do DNA , Estabilidade Enzimática , Temperatura Alta , Dados de Sequência Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Fases de Leitura Aberta , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica
17.
Biotechnol Bioeng ; 87(1): 99-103, 2004 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-15211493

RESUMO

Bio-Strand, Inc., has developed a novel DNA microarray platform utilizing a three-dimensional (3D) DNA format. DNA probes or polymerase chain reaction (PCR) products are spotted onto a thread-like scaffold, which is then wound onto a cylindrical core. By wrapping the thread around the core, high efficiencies are achieved in sample analysis. Using allele-specific oligo (ASO) competitive hybridization (with Cy5 fluorescently labeled sequences), hybridized arrays are visualized using a helium-neon (HeNe) laser and quantitated/scored. The method can readily detect single nucleotide differences. We demonstrate the use of this Bio-Strand 3D array in the analysis of a single nucleotide polymorphism (SNP).


Assuntos
Carbocianinas/química , Sondas de DNA/química , DNA/química , Nylons/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
18.
Biotechnol Bioeng ; 86(6): 667-71, 2004 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-15137078

RESUMO

We have developed an integrated automation system for genetic analysis and gene manipulation. The system, SX-8G Plus, is equipped with an 8-nozzle dispensing unit, a thermal cycler, a cooled reagent reservoir, four tip storage racks, four microplate platforms, buffer reservoirs, an agarose gel electrophoresis unit, a power supply, a pump for exchanging electrophoresis buffer, and a CCD camera. Automation of nucleic acid extraction and purification, the most difficult step in automating genetic analysis and gene manipulation, was realized using magnetic beads with Magtration Technology, which we have previously developed for automating the handling of paramagnetic beads. Using this system, we could perform the automated separation and purification of DNA fragments by agarose gel electrophoresis starting from sample loading. The system would enable the automation of almost all procedures in genetic analysis and gene manipulation.


Assuntos
DNA/isolamento & purificação , Engenharia Genética , Magnetismo/instrumentação , Microesferas , Análise de Sequência de DNA , Computadores , Eletroforese em Gel de Ágar , Desenho de Equipamento , Amplificação de Genes , Reação em Cadeia da Polimerase , Pyrococcus horikoshii/genética
19.
J Biotechnol ; 108(2): 153-9, 2004 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-15129724

RESUMO

A fully automated immunoassay of 17beta-estradiol (E2) was performed using anti-E2 monoclonal antibody immobilized on bacterial magnetic particles (AntiE2-BMPs) and alkaline phosphatase-conjugated E2 (ALP-E2). E2 concentration in environmental water samples was evaluated by decrease in luminescence based on competitive reaction. A linear correlation between the luminescence intensity and E2 concentration was obtained between 0.5 and 5 ppb. The minimum detectable concentration of E2 was 20 ppt. All measurement steps were done within 0.5 h. The analysis of environmental water samples by a commercially available ELISA kit and the BMP-based immunoassay gave good correlation plots with a correlation efficient of 0.992. These results suggest that the fully automated system using the BMP-based immunoassay has some advantages in the high rapidity and sensitivity of the measurement. This system will enable us to determine low E2 concentrations without sample condensation.


Assuntos
Proteínas de Bactérias/imunologia , Monitoramento Ambiental/métodos , Estradiol/análise , Estradiol/imunologia , Técnicas Imunoenzimáticas/métodos , Proteínas de Membrana/imunologia , Poluentes Químicos da Água/análise , Reprodutibilidade dos Testes , Robótica/métodos , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodos
20.
Pharmacogenomics ; 3(5): 697-708, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12223054

RESUMO

The majority of research for genome analysis has shifted from nucleic acid sequencing to the biological functional analysis of each gene. Based on past success, it may not be long before genome diagnostics becomes a widespread tool in human, veterinary and botany research fields. Genome analysis involves the processes of nucleic acid purification, amplification, labeling and signal detection (specific reaction, separation and signal counting). Except for the purification of nucleic acids, the other processes cannot be achieved without instruments, resulting in the advancement of automation processes. Since purification of nucleic acids can be done manually, automating this process has been delayed. However, because the purification of nucleic acids using magnetic particles is suitable for automation, its development has also been accelerated. The need for full automation for other processes is not as great because the majority of genome analysis is to identify the nucleic acid sequence and analyze genome expression. However, once useful diagnostic tools are generated, the desire for full automation will significantly increase. In order to develop realistic and practical automation, various technologies developed for each process in genome analysis have to be evaluated and only a few technologies, useful for automation, selected. The other key factor in automation is the development of methods to manage reagents and reaction mixtures precisely without any risks specifically related to genome handling, such as cross-contamination. Methods using magnetic particles, which have been used for the automation of nucleic acid purification and immunoassay, appear to be the most promising way to automate processes used in biological research.


Assuntos
Mapeamento Cromossômico/instrumentação , Magnetismo/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Mapeamento Cromossômico/métodos , Mapeamento Cromossômico/tendências , Genoma , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Imunoensaio/tendências , Técnicas de Amplificação de Ácido Nucleico/tendências , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/tendências
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