Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 670
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Anal Bioanal Chem ; 411(23): 6155-6163, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300857

RESUMO

Electrophoresis has demonstrated utility as tool for screening of small molecule modulators of protein-protein interactions and enzyme targets. Screening of large chemical libraries requires high-throughput separations. Such fast separation can be accessed by microchip electrophoresis. Here, microchip gel electrophoresis separations of proteins are achieved in 2.6 s with 1200 V/cm and 3-mm separation lengths. However, such fast separations can still suffer from limited overall throughput from sample introduction constraints. Automated introduction of microfluidic droplets has been demonstrated to overcome this limitation. Most devices for coupling microfluidic droplets to microchip electrophoresis are only compatible with free-solution separations. Here, we present a device that is compatible with coupling droplets to gel and free-solution electrophoresis. In this device, automated sample introduction is based on a novel mechanism of carrier phase separation using the difference in density of the carrier phase and the running buffer. This device is demonstrated for microchip gel electrophoresis and free-solution electrophoresis separations of protein-protein interaction and enzyme samples, respectively. Throughputs of about 10 s per sample are achieved and over 1000 separations are demonstrated without reconditioning of the device. Graphical abstract.


Assuntos
Eletroforese em Microchip/instrumentação , Mapeamento de Interação de Proteínas/instrumentação , Biocatálise , Desenho de Equipamento , Géis/química , Mapas de Interação de Proteínas , Proteínas/metabolismo
2.
Anal Sci ; 35(10): 1103-1109, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31231088

RESUMO

A dual-channel microchip electrophoresis (ME) with in-channel amperometric detection was developed for cefoperazone and sulbactam determination simultaneously. In this study, a microelectrode detector was made of gold nanoparticles (GNPs) modified indium tin oxide (ITO)-coated poly-ethylene terephthalate (PET) film. The parameters including detection potential applied on working electrode, buffer concentration and pH value were optimized to improve the detection sensitivity and separation efficiency of cefoperazone and sulbactam. Under the optimal conditions, sensitive detection of cefoperazone and sulbactam was obtained with limits of detection (LODs) (S/N = 3) of 0.52 and 0.75 µg/mL, respectively. The plasma sample, which was from a patient with a brain injury taking Sulperazone, was successfully detected with a simple sample pretreatment process by dual-channel ME amperometric detection. This rapid and sensitive method possesses practical potential in clinical applications, and could provide a guidance for clinical rational drug use.


Assuntos
Cefoperazona/análise , Eletroforese em Microchip/instrumentação , Sulbactam/análise , Métodos Analíticos de Preparação de Amostras , Tampões (Química) , Cefoperazona/sangue , Cefoperazona/química , Eletroquímica , Humanos , Concentração de Íons de Hidrogênio , Sulbactam/sangue , Sulbactam/química , Fatores de Tempo
3.
Electrophoresis ; 40(16-17): 2172-2179, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30953376

RESUMO

The fouling and stability are two most critical limiting factors for practical applications of surface-enhanced Raman scattering (SERS)-based microfluidic electrophoresis device. Herein, we present a novel biomimetic nanoengineering strategy to achieve a SERS substrate featuring antifouling ability, good stability, and reliable quantitative capability. Typically, by employing tea polyphenol as the reducing agent, the substrate made of silver core-gold shell nanostructures in situ grown on silicon wafer surface is fabricated. The core-shell nanostructures are further embedded with internal standard molecules. Remarkably, the fabricated substrate preserves distinct SERS effects, adaptable reproducibility, and reliable quantitative ability even if the substrate is incubated with 15% H2 O2 , 13% HNO3 , or 108  CFU/mL bacteria, or suffered from 12-day continuous vibration at 250 rpm/min in PBS buffer. As a proof-of-concept application, the DNA-functionalized substrate is capable of precise quantification of Hg2+ with a limit of detection down to ca. 1 pM even in sewage water.


Assuntos
Biomimética/métodos , Nanopartículas Metálicas/química , Prata , Análise Espectral Raman/instrumentação , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Eletroforese em Microchip/instrumentação , Contaminação de Equipamentos , Desenho de Equipamento , Limite de Detecção , Modelos Lineares , Mercúrio/análise , Reprodutibilidade dos Testes , Esgotos/química , Prata/química , Prata/farmacologia
4.
Electrophoresis ; 40(16-17): 2165-2171, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30861170

RESUMO

Micro free flow electrophoresis (µFFE) is a valuable technique capable of high throughput rapid microscale electrophoretic separation along with mild operating conditions. However, the stream flow separation nature of free flow electrophoresis affects its separation performance with additional stream broadening due to sample stream deflection. To reduce stream broadening and enhance separation performance of µFFE, we presented a simple microfluidic device that enables injection bandwidth control. A pinched injection was formed in the reported µFFE system using operating buffer at sample flow rate ratio (r) setting. Initial bandwidth at the entrance of separation chamber can be shrunk from 800 to 30 µm when r increased from 1 to 256. Stream broadening at the exit of separation chamber can be reduced by about 96% when r increased from 4 to 128, according to both theoretical and experimental results. Moreover, the separation resolution for a dye mixture was enhanced by a factor of 4 when r increased from 16 to 128, which corresponded to an 80% reduction in sample initial bandwidth. Furthermore, a similar enhancement on amino acids separation was obtained by using injection control in the reported µFFE device and readily integrated into online/offline sample preparation and/or downstream analysis procedures.


Assuntos
Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Aminoácidos/análise , Aminoácidos/isolamento & purificação , Corantes/análise , Corantes/isolamento & purificação , Desenho de Equipamento , Modelos Químicos
5.
Methods Mol Biol ; 1972: 175-184, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30847791

RESUMO

An unmet need exists for a clinical diagnostic to determine preterm birth (PTB) risk. Such an assessment is possible with high sensitivity and specificity using a panel of nine biomarkers. An integrated microfluidic analysis system for these biomarkers is being developed which includes microchip electrophoresis (µCE) separation. A t-shaped microchip device can be used to test the µCE portion of this integrated system to find appropriate separation conditions. These t-shaped microchips can be fabricated using photolithographically patterned Si templates and hot embossing. PTB biomarkers can be fluorescently labeled using an amine-reactive dye prior to µCE. The µCE conditions established using this t-shaped device should be useful in developing a complete integrated microfluidic system for PTB risk assessment.


Assuntos
Biomarcadores/análise , Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Corantes Fluorescentes/química , Nascimento Prematuro/diagnóstico , Eletrodos , Humanos , Silício/química
6.
Electrophoresis ; 40(9): 1322-1330, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30657598

RESUMO

The fabrication of PDMS microfluidic structures through soft lithography is widely reported. While this well-established method gives high precision microstructures and has been successfully used for many researchers, it often requires sophisticated instrumentation and expensive materials such as clean room facilities and photoresists. Thus, we present here a simple protocol that allows the rapid molding of simple linear microchannels in PDMS substrates aiming microfluidics-based applications. It might serve as an alternative to researchers that do not have access to sophisticated facilities such as clean rooms. The method developed here consists on the use of pencil graphite leads as template for the molding of PDMS channels. It yields structures that can be used for several applications, such as housing support for electrochemical sensors or channels for flow devices. Here, the microdevices produced through this protocol were employed for the accommodation of carbon black paste, which was utilized for the first time as amperometric sensor in microchip electrophoresis. This platform was successfully used for the separation and detection of model analytes. Ascorbic acid and iodide were separated within 45 s with peak resolution of 1.2 and sensitivities of 198 and 492 pA/µM, respectively. The background noise was ca. 84 pA. The analytical usefulness of the system developed was successfully tested through the quantification of iodide in commercial pharmaceutical formulations. It demonstrates good efficiency of the microfabrication protocol developed and enables its use for the easy and rapid prototyping of PDMS structures over a low fabrication cost.


Assuntos
Microfluídica/instrumentação , Dimetilpolisiloxanos , Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Desenho de Equipamento , Grafite , Microfluídica/economia
7.
Electrophoresis ; 40(18-19): 2478-2483, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30637781

RESUMO

In this study, we found that the polarity switching was effective to enrich and separate fluorescent analytes which have weakly-dissociated groups in a floating platinum electrode (width, 50 µm; thickness, 2.5 µm)-integrated straight-channel in microchip electrophoresis (MCE). In the straight channel filled with an Alexa Flour 488 (AF488) solution, a sharp peak was observed after the polarity inversion with a 530-fold enhancement of the sensitivity relative to the conventional MCE analysis. By using a fluorescent pH indicator, we verified that a sharp high-pH zone was generated nearby the floating electrode and moved toward the anode with maintaining the high pH, which induced the sample enrichment like a dynamic pH junction mechanism. In the floating electrode-embedded channel, the mixture of AF488-labeled proteins was also well concentrated and separated within 100 s.


Assuntos
Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Eletrodos , Desenho de Equipamento , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Proteínas/análise , Proteínas/química , Proteínas/isolamento & purificação , Reprodutibilidade dos Testes
8.
Methods Mol Biol ; 1906: 65-78, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30488385

RESUMO

Electrophoretic on-line sample preconcentration techniques in microfluidic channels improve the sensitivity prior to the separation. Among various techniques, the most important field-amplified sample stacking and sweeping on cross-channel microchips are demonstrated. As a novel microfluidic preconcentration approach, a large-volume sample stacking with electroosmotic flow pump (LVSEP) on straight-channel chips is also presented, which can omit a complicated voltage program for sample injection processes. In this chapter, we describe how to prepare and how to run these on-line sample preconcentration methods in microchip electrophoresis.


Assuntos
Eletro-Osmose/instrumentação , Eletroforese em Microchip/métodos , Tampões (Química) , Calibragem , Eletro-Osmose/métodos , Eletroforese em Microchip/instrumentação , Limite de Detecção , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
9.
Methods Mol Biol ; 1906: 79-85, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30488386

RESUMO

Microchip electrophoresis is a versatile separation technique. Electrochemical detection is suitable to apply to microdevices due to its easy integration to the fabrication process and good sensitivity and selectivity. Here we describe the procedures to prepare Pt band electrodes deposited on glass to couple to polydimethylsiloxane (PDMS) microchips aiming the separation and detection of nitrite using an isolated potentiostat.


Assuntos
Dimetilpolisiloxanos/química , Eletroforese em Microchip/instrumentação , Nitritos/análise , Técnicas Biossensoriais/instrumentação , Eletrodos , Eletroforese em Microchip/métodos , Vidro
10.
Methods Mol Biol ; 1906: 113-124, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30488389

RESUMO

Free-flow electrophoresis (FFE) may be used for continuous and preparative separation of a wide variety of biomolecules. Isoelectric focusing (IEF) provides for the separation of compounds according to their isoelectric point (pI). Here we describe a microfluidic chip-based protocol for the fabrication, application, and optical monitoring of free-flow isoelectric focusing (FFIEF) of proteins and peptides on the microscale with optical surveillance of the microscopic pH gradient provided by an integrated pH sensing layer. This protocol may be used with modifications also for the FFIEF of other biomolecules and may serve as template for the fabrication of microfluidic chips with integrated fluorescent or luminescent pH sensor layers for FFE and other applications.


Assuntos
Eletroforese em Microchip/métodos , Focalização Isoelétrica/métodos , Peptídeos/análise , Proteínas/análise , Técnicas Biossensoriais , Eletroforese em Microchip/instrumentação , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/instrumentação , Microscopia de Fluorescência , Miniaturização
11.
Methods Mol Biol ; 1906: 125-132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30488390

RESUMO

Gradient-based electrophoretic separations enable simultaneous separation and concentration of molecules. Compared with conventional injection-based separations, they enable enrichment of low-concentration analytes from larger sample volumes that are not limited by an injection volume. We have demonstrated that a nanochannel, connecting two chemically different reservoirs, can maintain a stationary chemical gradient while trapping biomolecules and effectively averaging out many of the complex physicochemical hydrodynamics that would broaden the bands in a meso- or microscale capillary. Here we describe chemical and physical methods that enable this work.


Assuntos
Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Condutividade Elétrica , Desenho de Equipamento , Força Próton-Motriz
12.
Methods Mol Biol ; 1906: 133-142, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30488391

RESUMO

This chapter describes the development of paper-based microchip electrophoresis (pME) devices for the separation of clinically relevant compounds. pME were fabricated by laser cut and thermal lamination process using polyester pouches. In addition, hand-drawn pencil electrodes were integrated to the device to perform capacitively coupled contactless conductivity detection (C4D). Finished device costs less than US$ 0.10 and did not require either sophisticated instrumentation or clean room facilities. Furthermore, pME is lightweight, easy to handle, flexible, and robust. pME-C4D device revealed an excellent capacity to separate BSA and creatinine in less than 150 s with baseline resolution. The device proposed in this chapter has proven to be a good alternative as a platform for the diagnosis of diseases from renal disorders such as diabetes mellitus and heart disease.


Assuntos
Creatinina/análise , Eletroforese em Microchip/instrumentação , Desenho de Equipamento/métodos , Soroalbumina Bovina/análise , Animais , Bovinos , Diabetes Mellitus/diagnóstico , Condutividade Elétrica , Eletroforese em Microchip/métodos , Cardiopatias/diagnóstico , Humanos , Nefropatias/diagnóstico , Lasers , Papel
13.
Methods Mol Biol ; 1906: 197-206, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30488394

RESUMO

Microchip electrophoresis (ME) results from miniaturization of capillary electrophoresis (CE) to a microfabricated separation device. Both techniques have common characteristics, but in some aspects, the microfluidic separation device has unique features resulting from its planar miniaturized format. Here we describe the process to transfer of CE to ME and the benefits and drawbacks of the chip with respect to the capillary. A practical guide for method development on the microchip for small ionizable molecules such as phenolic compounds, amino acids, or alkaloids is also presented.


Assuntos
Eletroforese em Microchip/instrumentação , Bibliotecas de Moléculas Pequenas/análise , Alcaloides/análise , Aminoácidos/análise , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Fenóis/análise
14.
Methods Mol Biol ; 1906: 225-237, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30488396

RESUMO

The past two decades have witnessed remarkable advances in the development of microfluidic devices as bioanalytical platforms for the analysis of biological molecules. The implementation of mass spectrometry (MS) detection systems on these devices has become inevitable, and various chip-MS ionization interfaces have been developed. As electrospray ionization (ESI) is particularly relevant for the analysis of large biological molecules such as proteins or peptides, efforts have focused on advancing interfaces that meet the demands of nano-separation techniques that are typically used prior to MS detection. Achieving stable ESI conditions that enable sensitive MS detection is, however, not trivial, especially when the spray is generated from a microfabricated platform. This chapter is aimed at providing a step-by-step protocol for producing stable and efficient electrospray sample ionization from microfluidic chips that are used for capillary electrophoresis (CE) separations.


Assuntos
Eletroforese em Microchip/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Eletroforese Capilar/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação
15.
Methods Mol Biol ; 1906: 239-249, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30488397

RESUMO

It is well known that the resolving power of capillary zone electrophoretic separations may be improved with an increase in the applied electric field strength and separation time. While large electric fields may be realized in short analysis channels commonly employed in microfluidic systems, this experimental design is not suitable for achieving long separation times. In this chapter, we describe the use of a steady and/or a periodic pressure-driven backflow to increase the separation time in short microchannels thereby enabling the analysis of closely related species on microchip devices. The reported backflow was realized in our assays using an on-chip pressure-generation capability that relied on the partial blockage of electroosmotic flow around a junction of two glass channel segments having different depths. Although the noted strategy led to additional band broadening in the system, the resolving power of our device was observed to substantially improve upon introduction of the reported steady/periodic pressure-driven backflow for analysis channels shallower than 5 µm.


Assuntos
Eletroforese em Microchip/métodos , Eletro-Osmose , Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Eletroforese em Microchip/instrumentação , Desenho de Equipamento , Concentração de Íons de Hidrogênio , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
16.
Methods Mol Biol ; 1855: 327-340, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30426429

RESUMO

Aggregation of beta-amyloid peptides especially Aß1-42 in amyloid plaques is one of the major neuropathological events in Alzheimer's disease. This event is normally accompanied by a relative reduction of the concentration of Aß1-42 in the cerebrospinal fluid (CSF) of patient developing the signs of Alzheimer's disease. Here, we describe methods for isolation and for microchip gel electrophoresis of Aß peptides in polydimethylsiloxane (PDMS) microfluidic chip. The method was applied to compare the relative concentration of Aß1-42 with other Aß peptides, for example, Aß 1-40 in CSF. In order to increase the sensitivity of detection, Aß peptides in the CSF samples were first captured and concentrated using magnetic beads coated with specific anti-Aß antibodies.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Eletroforese em Microchip/métodos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Peptídeos beta-Amiloides/isolamento & purificação , Anticorpos Imobilizados/química , Dimetilpolisiloxanos/química , Eletroforese em Microchip/instrumentação , Desenho de Equipamento , Humanos , Imãs/química , Fragmentos de Peptídeos/isolamento & purificação
17.
Electrophoresis ; 40(3): 455-461, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30450561

RESUMO

A new multi-stacking pre-concentration procedure based on field-enhanced sample injection (FESI), field-amplified sample stacking, and transient isotachophoresis was developed and implemented in a compact microchip electrophoresis (MCE) with a double T-junction glass chip, coupled with an on-chip capacitively coupled contactless conductivity detection (C4 D) system. A mixture of the cationic target analyte and the terminating electrolyte (TE) from the two sample reservoirs was injected under FESI conditions within the two sample-loading channels. At the double T-junction, the stacked analyte zones were further concentrated under field-amplified stacking conditions and then subsequently focused by transient-isotachophoresis and separated along the separation channels. The proposed multi-stacking strategy was verified under a Universal Serial Bus (USB) fluorescence microscope employing Rhodamine 6G as the model analyte. This developed approach was subsequently used to monitor the target quinine present in human plasma samples. The total analysis time for quinine was approximately 200 s with a sensitivity enhancement factor of approximately 61 when compared to the typical gated injection. The detection and quantification limits of the developed approach for quinine were 3.0 µg/mL and 10 µg/mL, respectively, with intraday and interday repeatability (%RSDs, n = 5) of 3.6 and 4.4%. Recoveries in spiked human plasma were 98.1-99.8%.


Assuntos
Análise Química do Sangue/instrumentação , Eletroforese em Microchip/instrumentação , Quinina/sangue , Análise Química do Sangue/métodos , Eletroforese em Microchip/métodos , Desenho de Equipamento , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
18.
Biosens Bioelectron ; 122: 263-283, 2018 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-30268964

RESUMO

Antibiotics are a type of antimicrobial drug with the ubiquitous presence in foodstuff that effectively applied to treat the diseases and promote the animal growth worldwide. Chloramphenicol as one of the antibiotics with the broad action spectrum against Gram-positive and Gram-negative bacteria is widely applied for the effective treatment of infectious diseases in humans and animals. Unfortunately, the serious side effects of chloramphenicol, such as aplastic anemia, kidney damage, nausea, and diarrhea restrict its application in foodstuff and biomedical fields. Development of the sufficiently sensitive methods to detect chloramphenicol residues in food and clinical diagnosis seems to be an essential demand. Biosensors have been introduced as the promising tools to overcome the requirement. As one of the newest types of the biosensors, aptamer-based biosensors (aptasensors) are the efficient sensing platforms for the chloramphenicol monitoring. In the present review, we summarize the recent achievements of the accessible aptasensors for qualitative detection and quantitative determination of chloramphenicol as a candidate of the antibiotics. The present chloramphenicol aptasensors can be classified in two main optical and electrochemical categories. Also, the other formats of the aptasensing assays like the high performance liquid chromatography (HPLC) and microchip electrophoresis (MCE) have been reviewed. The enormous interest in utilizing the diverse nanomaterials is also highlighted in the fabrication of the chloramphenicol aptasensors. Finally, some results are presented based on the advantages and disadvantages of the studied aptasensors to achieve a promising perspective for designing the novel antibiotics test kits.


Assuntos
Antibacterianos/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Cloranfenicol/análise , Animais , Técnicas Biossensoriais/instrumentação , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Colorimetria/instrumentação , Colorimetria/métodos , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Desenho de Equipamento , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Humanos , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos
19.
Anal Chem ; 90(21): 13000-13006, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30335366

RESUMO

Improvements were made to a previously developed platform coupling microchip capillary electrophoresis (CE) with high pressure mass spectrometry (HPMS). The RF drive frequency was increased to over 30 MHz from less than 10 MHz, and the ion trap was scaled down to 100 µm critical dimensions. A stretched length ion trap was used to improve sensitivity, and a tube lens was used to improve ion transmission. Detection of the 20 common amino acids was demonstrated, resulting in an average improvement of signal-to-noise of 28 times and an average improvement in peak width of 2.6 times over those obtained in previous work. Consumption of amino acids by cells in growth media was monitored over time using the improved CE-HPMS platform, and several amino acids were shown to be consumed at different rates, demonstrating the potential for real-time bioreactor monitoring.


Assuntos
Eletroforese em Microchip/instrumentação , Escherichia coli K12/crescimento & desenvolvimento , Dispositivos Lab-On-A-Chip , Aminoácidos/análise , Eletroforese Capilar/métodos , Eletroforese em Microchip/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos
20.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1097-1098: 10-17, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30195071

RESUMO

Glutathione S-transferase (GST) polymorphism (M1 = 215 bp and T1 = 480 bp) can cause liver damage and increase the risk of cancer. In this study, voltage programming (VP)-based microchip electrophoresis (ME) with a laser-induced fluorescence (LIF) detector was developed to detect specific sizes of DNA fragments. The optimum conditions for a single-channel microchip were as follows: 4 kV for 0-9.5 s, 1.5 kV for 9.5-15.5 s, and 4 kV for 15.5-30 s. Next, these conditions were applied to another microchip that was constructed with many channels making possible simultaneous parallel detection. Finally, GST genes extracted from human blood were amplified by polymerase chain reaction (PCR) and were introduced into the multi-channel microchip. Target DNA molecules amplified by only 10 PCR cycles could be detected by the VP-based multi-channel ME method, but not by slab gel electrophoresis (SGE). In addition, the migration time for ME was <15 s, which was 700 times faster than conventional SGE. The developed VP-based multi-channel ME method with LIF detection was demonstrated to be an effective, rapid analysis technique for highly sensitive and high-throughput screening of GST genes.


Assuntos
Eletroforese em Microchip/métodos , Glutationa Transferase/genética , Polimorfismo Genético/genética , Análise de Sequência de DNA/métodos , DNA/análise , DNA/genética , Eletroforese em Microchip/instrumentação , Desenho de Equipamento , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/instrumentação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA