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1.
Mikrochim Acta ; 187(2): 144, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31970520

RESUMO

Iron(III-immobilized magnetic nano-composites (MNCs) were first fabricated using one-step aqueous self-assembly of oligopeptides (Glu-Pro-Ala-Lys-Ala-Lys-Ala-Lys; EPAK-VI) for the highly selective capture of phosphopeptides from complex biological samples. Under physiological conditions, EPAK-VI can readily self-organize into a robust and complete coating layer mainly composed of ß-sheets and ß-turns on the surface of Fe3O4@GO and Fe3O4@C MNCs. Tailored by the cyclic structure of proline, the Glu-Pro motifs of EPAK-VI are vertically erected on the surface and thus serve as an effective linker to chelate Fe3+ through carboxyl (COO-) group in the glutamic acid (E) residues. The ionic hydrogen bonds between the ε-amino groups and the surface negative charges coupled with intermolecular hydrogen bonds render the EPAK-VI coating on the MNCs insusceptible to repeated extreme washing conditions. The Fe3+-EPAK-VI coated MNCs exhibit high enrichment efficiency for ß-casein tryptic digest (0.05 fmol µL-1), excellent selectivity from mixed digests (ß-casein/bovine serum albumin, mass ratio 1:500), and high recovery rate (over 80%). Graphical abstractSchematic representation of the fabrication of Fe3+-immobilized MNCs for phosphopeptide enrichment.

2.
J Hazard Mater ; 382: 121113, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31479827

RESUMO

Copper ion (Cu (II)) pollution has attracted much attention due to its remarkable toxic domino effect at excess amount. Efficient Cu (II) ions removal is thus a prerequisite for wastewater recycling. Herein, we present a facile and environmentally benign strategy to fabricate thiol (SH)-functionalized Fe3O4@C nanoparticles (denoted as Fe3O4@C-SH NPs) based on one-step self-assembling of a bifunctional oligopeptide with a sequence of Cys-Lys-Cys-Lys-Cys-Lys (CK-VI) for highly efficient removal of copper ions (Cu (II)) in aqueous solutions. Under the physiological conditions, CK-VI readily self-organized into a robust and tailor-made functional monolayer predominately composed of well-packed ß-sheets on the surface of Fe3O4@C NPs with their thiol groups standing on the outermost layer. The resulting Fe3O4@C-SH NPs containing abundant thiol active sites exhibited excellent adsorption capacity (up to 28.8 mg g-1) and selectivity for Cu (II) ions over coexisting ions. Compared with other covalent grafting methods with multistep processes and in harsh conditions, the proposed oligopeptides assembly-based coating method makes it possible to rapidly fabricate the Fe3O4@C-SH NPs in a simple mild one-step aqueous process with low cost. The current study provides facile and environmentally friendly approaches to rapidly tailor multifunctional surfaces of NPs for various toxic metal ions removal from wastewater.

3.
Anal Chim Acta ; 1088: 63-71, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31623717

RESUMO

Magnetic nanoparticles (MNPs) have been widely explored in enrichment of low-abundance glycoproteins/glycopeptides prior to mass spectrometry analysis in glycoproteomics. Currently, most functional groups for recognizing glycoproteins/glycopeptides are usually immobilized on the nanomaterial surface based on covalent modification, which suffers from multistep treatment, surface-dependence, and harsh conditions. In this work, we first report a facile and rapid method for surface functionalization and subsequent glycopeptides enrichment via one-step assembly of maltose-modified oligopeptides with a sequence of Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys (AEK8-maltose). In physiological conditions, AEK8-maltose readily self-organized into a complete coating layer dominated by ß-sheets on the surface of SiO2@Fe3O4 and C@Fe3O4 MNPs, which remain intact to repeat washing with acidic organic and aqueous solutions extensively used in the sample enrichment treatments. Thus, the resulting AEK8-maltose functionalized MNPs show excellent performance in enrichment of glycopeptides in standard glycoprotein digests (24 glycopeptides from horseradish peroxidase (HRP), 31 glycopeptides from immunoglobulin (IgG)) and human serum digests (282 glycopeptides), including rapid enrichment speed (5 min), high detection sensitivity (0.001 ng/µL HRP), high selectivity (mass ratios of HRP and bovine serum albumin (BSA) digests up to 1:150), good enrichment recovery (over 86.3%), remarkable stability (repeatable for more than 8 times), and excellent renewability, which are better than or comparable with the literature results reported to date. The current work based on self-assembling oligopeptides provides a mild, economic and nontoxic procedure for one-step surface functionalization of various nanomaterials.

4.
Anal Chem ; 90(22): 13708-13713, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30350952

RESUMO

Adenosine triphosphate (ATP) as a primary energy source plays a unique role in the regulation of all cellular events. The necessity to detect ATP requires sensitive and accurate quantitative analytical strategies. Herein, we present our study of developing a MoS2 nanosheet-enhanced aptasensor for fluorescence polarization-based ATP detection. A bifunctional DNA strand was designed to consist of chimeric aptamers that recognize and capture ATP and berberine, a fluorescence enhancer. In the absence of ATP, the DNA strand bound to berberine will be hydrolyzed when Exonuclease I (Exo I) is introduced, releasing berberine as a result. In contrast, when ATP is present, ATP aptamer folds into a G-quadruplex structure; thus, the complex can resist degradation by Exo I to maintain berberine for fluorescent detection purpose. In addition, to magnify the fluorescence polarization (FP) signal, MoS2 nanosheets were also adopted in the system. This nanosheets-enhanced FP strategy is simple and facile which does not require traditional dye-labeled DNA strands and complex operation steps. The developed fluorescence polarization aptasensor showed high sensitivity for the quantification of ATP with a detection limit of 34.4 nM, performing well both in buffer solution and in biological samples.


Assuntos
Trifosfato de Adenosina/análise , Aptâmeros de Nucleotídeos/química , Polarização de Fluorescência/métodos , Berberina/química , Limite de Detecção , Difração de Pó
5.
Talanta ; 188: 708-713, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30029436

RESUMO

A novel ratiometric fluorescence sensing system based on cholesterol oxidase-functionalized dual-color mesoporous silica nanoparticles (MSNs)@metal-organic framework core-shell nanocomposite is demonstrated for cholesterol detection. MSNs were first loaded with 5-aminofluorescein (AF) inside pores and then wrapped with red-emission CdTe quantum dots (QDs) on the surface to seal in the dye molecules, forming the signal displaying unit (AF-MSN-QDs). Next, AF-MSN-QDs were encapsulated with zeolitic imidazolate framework (ZIF-8) to form a transition layer with distinct size-selectivity, which not only protected the cores from corrosion but also greatly decreased background interference from large molecules. More significantly, the ZIF-8 shells showed high affinity for most enzymes, which made it possible for cholesterol oxidase (ChOx) to self-organize on the surface of ZIF-8-encapsulated AF-MSN-QDs via chemo-physical adsorption, forming novel core-shell nanocomposites (AF-MSN-QD@ZIF-8-ChOx) as a sensing platform for cholesterol detection. The detectable signal was monitored by enzymatic product-quenching fluorescence of the QDs. The fluorescence changes of I520/I618 showed excellent linearity with H2O2 concentrations in the range of 5-100 nM, with a limit of detection (LOD) as low as 0.89 nM. As a proof-of-concept, cholesterol was selectively detected with beneficial LOD as low as 0.923 µg/mL, demonstrating the great potential of this biosensor platform for other biologically important molecules with H2O2-producing oxidases.


Assuntos
Colesterol Oxidase/química , Colesterol/análise , Corantes Fluorescentes/química , Nanocompostos/química , Nanopartículas/química , Dióxido de Silício/química , Adsorção , Técnicas Biossensoriais/métodos , Cádmio/química , Colesterol/metabolismo , Fluoresceínas/química , Fluorescência , Peróxido de Hidrogênio/síntese química , Imidazóis/química , Limite de Detecção , Pontos Quânticos/química , Telúrio/química , Zeolitas/química
6.
Talanta ; 179: 531-537, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29310271

RESUMO

Highly selective and sensitive detection methods are very important for the early diagnosis of prostate-specific antigen (PSA). Here, we present a novel peptide/Fe3O4@SiO2-Au nanocomposite-based fluorescence biosensor for highly selective and sensitive detection of PSA. The biosensor was made by self-organizing 5-FAM labeled peptides onto the surface of magnetic Fe3O4@SiO2-Au nanocomposites (MNCPs), resulting in efficient quenching of the FAM fluorescence. The PSA specifically recognized and cleaved the 5-FAM-labeled peptides, leading to the fluorescence recovery. This is the first report of the MNCPs by in situ growth of Au nanoparticles (AuNPs) on the SiO2 encapsulated single Fe3O4 nanocubes. The MNCPs feature robust salt stability, and allow for effective fluorescence quenching and easy magnetic separation, which greatly decrease the background fluorescence. The peptide/MNCPs-based fluorescence biosensor measure a wide range of concentrations of PSA, from 1.0 × 10-12 to 1.0 × 10-9g/mL, with a limit of detection (LOD) of 3.0 × 10-13g/mL in both standard solutions and serum samples, demonstrating the great potential of this biosensor platform for use in clinical and biological assays.


Assuntos
Técnicas Biossensoriais/métodos , Recuperação de Fluorescência Após Fotodegradação/métodos , Nanopartículas de Magnetita/química , Nanocompostos/química , Peptídeos/química , Antígeno Prostático Específico/sangue , Óxido Ferroso-Férrico/química , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Nanopartículas de Magnetita/ultraestrutura , Masculino , Nanocompostos/ultraestrutura , Próstata/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Dióxido de Silício/química , Coloração e Rotulagem/métodos
7.
Anal Chim Acta ; 998: 60-66, 2018 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-29153087

RESUMO

In the present study, a facile fluorescence aptasensor based on two-dimensional sheet metal-organic frameworks of N,N-bis(2-hydroxyethyl)dithiooxamidato copper(II) (H2dtoaCu) was developed for the sensitive detection of adenosine triphosphate (ATP). The sensing mechanism was based on the noncovalent interaction between FAM-labeled (fluorescein amidite) ATP aptamers and H2dtoaCu. In the absence of ATP, the FAM-labeled aptamer readily adsorbs onto H2dtoaCu, mainly via π-π stacking and hydrogen bond interactions between the nucleotide bases and the H2dtoaCu surface, leading to the reduction of fluorescence intensity of the FAM by photoinduced electron transfer (PET). In the presence of ATP, the FAM-labeled aptamer specifically forms ATP-binding aptamer complexes which exhibit only weak adsorption on the H2dtoaCu surface. Thus, the fluorescence of the FAM-labeled ATP aptamer remained largely unchanged. The fluorescence aptasensor exhibited a good linear relationship between the fluorescence intensity and the logarithm concentration of ATP over a range of 25-400 nM, with a detection limit of 8.19 nM (3S/N). ATP analogs such as guanosine triphosphate, uridine triphosphate, and cytidine triphosphate have negligible effect on the aptasensor performance due to the high selectivity of the ATP aptamer to its target, showing promising potential in real sample analysis.


Assuntos
Trifosfato de Adenosina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Fluorescência , Estruturas Metalorgânicas/química , Espectroscopia de Infravermelho com Transformada de Fourier
8.
Biosens Bioelectron ; 91: 328-333, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28040665

RESUMO

5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 ß-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues.


Assuntos
5-Metilcitosina/análogos & derivados , Ácidos Bóricos/química , DNA/química , Corantes Fluorescentes/química , Ácidos Polimetacrílicos/química , Espectrometria de Fluorescência/métodos , 5-Metilcitosina/análise , Animais , Química Encefálica , Fígado/química , Camundongos , Miocárdio/química , Quinolinas/química
9.
Biosens Bioelectron ; 87: 339-344, 2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27573301

RESUMO

In this work, we proposed a novel and facile method to monitor oxidase activities based on size-selective fluorescent quantum dot (QD)@metal-organic framework (MOF) core-shell nanocomposites (CSNCPs). The CSNCPs were synthesized from ZIF-8 and CdTe QDs in aqueous solution in 40min at room temperature with stirring. The prepared CdTe@ZIF-8 CSNCPs , which have excellent water dispersibility and stability, displays distinct fluorescence responses to hole scavengers of different molecular sizes (e.g., H2O2, substrate, and oxidase) due to the aperture limitation of the ZIF-8 shell. H2O2 can efficiently quench the fluorescence of CdTe@ZIF-8 CSNCPs over a linearity range of 1-100nM with a detection limit of 0.29nM, whereas large molecules such as substrate and oxidase have very little effect on its fluorescence. Therefore, the highly sensitive detection of oxidase activities was achieved by monitoring the fluorescence quenching of CdTe@ZIF-8 CSNCPs by H2O2 produced in the presence of substrate and oxidase, which is proportional to the oxidase activities. The linearity ranges of the uricase and glucose oxidase activity are 0.1-50U/L and 1-100U/L, respectively, and their detection limits are 0.024U/L and 0.26U/L, respectively. Therefore, the current QD@MOF CSNCPs based sensing system is a promising, widely applicable means of monitoring oxidase activities in biochemical research.


Assuntos
Compostos de Cádmio/química , Ensaios Enzimáticos/métodos , Glucose Oxidase/metabolismo , Nanocompostos/química , Compostos Organometálicos/química , Telúrio/química , Urato Oxidase/metabolismo , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Glucose Oxidase/análise , Limite de Detecção , Nanocompostos/ultraestrutura , Pontos Quânticos/química , Pontos Quânticos/ultraestrutura , Espectrometria de Fluorescência/métodos , Urato Oxidase/análise
10.
J Chromatogr A ; 1481: 152-157, 2017 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-28017563

RESUMO

A facile and efficient dynamic coating method using an ionic complementary peptide was established for high-performance separation of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled carbohydrates in a hybrid poly(dimethylsiloxane) (PDMS)/glass microfluidic channel. EAK16-II with a sequence of [(Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)2] can readily self-organize into a complete coating layer tightly adsorbed on both hydrophobic PDMS and hydrophilic glass surfaces, which efficiently suppressed nonspecific analyte adsorption and minimized electroosmotic flow (EOF). Separation conditions were systematically investigated with respect to EAK16-II concentration, running buffer, buffer pH, and field strength (Esep). Under the optimal conditions, rapid and reproducible separations of maltodextrin ladder, glycans from glucosamine capsules, tablets, and pomegranate peel extracts were achieved with over 450000 theoretical plates per meter in the hybrid PDMS/glass microchannels dynamically coated with 1.0mg/mL EAK16-II-0.05% n-dodecyl ß-d-maltoside (DDM), and the relative standard deviation (RSD) values were less than 3.2% (n=4) for the migration times. The present work provides a facile and efficient means to minimize EOF and nonspecific analyte adsorption in microfluidic chips fabricated in various substrates, thereby broadening the applications of microfluidic chips in complicated biological assays.


Assuntos
Carboidratos/análise , Dimetilpolisiloxanos/química , Vidro/química , Peptídeos/química , Adsorção , Tampões (Química) , Eletricidade , Eletro-Osmose , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Íons , Microfluídica , Microscopia de Força Atômica , Polissacarídeos/análise , Silanos/química , Propriedades de Superfície , Água/química
11.
Anal Chem ; 87(21): 11078-83, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26447651

RESUMO

Studying ligand-biomacromolecule interactions provides opportunities for creating new compounds that can efficiently regulate specific biological processes. Ribonucleic acid (RNA) molecules have become attractive drug targets since the discovery of their roles in modulating gene expression, while only a limited number of studies have investigated interactions between ligands and functional RNA molecules, especially those based on nanotechnology. DNA-protected silver nanoclusters (AgNCs) were used to investigate ligand-RNA interactions for the first time in this study. The anthracycline anticancer drug mitoxantrone (MTX) was found to quench the fluorescence of AgNCs. After adding human immunodeficiency virus trans-activation responsive region (TAR) RNA or Rev-response element (RRE) RNA to AgNCs-MTX mixtures, the fluorescence of the AgNCs recovered due to interactions between MTX with RNAs. The binding constants and number of binding sites of MTX to TAR and RRE RNA were determined through theoretical calculations. MTX-RNA interactions were further confirmed in fluorescence polarization and mass spectrometry experiments. The mechanism of MTX-based fluorescence quenching of the AgNCs was also explored. This study provides a new strategy for ligand-RNA binding interaction assay.


Assuntos
DNA/química , HIV/genética , Nanoestruturas , RNA Viral/química , Prata/química , Dicroísmo Circular , Fluorescência , Ligantes , Mitoxantrona/química
12.
Langmuir ; 31(21): 5891-8, 2015 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-25966872

RESUMO

Poly(dimethylsiloxane) (PDMS) has become a widely used material for microfluidic and biological applications. However, PDMS has unacceptably high levels of nonspecific protein adsorption, which significantly lowers the performance of PDMS-based microfluidic chips. Most existing methods to reduce protein fouling of PDMS are to make the surface more hydrophilic by surface oxidization, polymer grafting, and physisorbed coatings. These methods suffer from the relatively short-term stability, the multistep complex treatment procedure, or the insufficient adsorption reduction. Herein, we developed a novel and facile modification method based on self-assembled peptides with well-tailored amino acid composition and sequence, which can also interact strongly with the PDMS surface in the same way as proteins, for suppressing the nonspecific protein fouling and improving the biocompatibility of PDMS-based microfluidic chips. We first demonstrated that an ionic complementary peptide, EAR16-II with a sequence of [(Ala-Glu-Ala-Glu-Ala-Arg-Ala-Arg)2], can readily self-assemble into an amphipathic film predominantly composed of tightly packed ß-sheets on the native hydrophobic and plasma-oxidized hydrophilic PDMS surfaces upon low concentrations of carbohydrates. The self-assembled EAR16-II amphipathic film exposed its hydrophobic side to the solution and thus rendered the PDMS surface hydrophobic with water contact angles (WCAs) of around 110.0°. However, the self-assembled EAR16-II amphipathic film exhibited excellent protein-repelling and blood compatibility properties comparable to or better than those obtained with previously reported methods. A schematic model has been proposed to explain the interactions of EAR16-II with the PDMS surface and the antifouling capability of EAR16-II coatings at a molecular level. The current work will pave the way to the development of novel coating materials to address the nonspecific protein adsorption on PDMS, thereby broadening the potential uses of PDMS-based microfluidic chips in complex biological analysis.


Assuntos
Dimetilpolisiloxanos/química , Proteínas/química , Adsorção , Interações Hidrofóbicas e Hidrofílicas , Microfluídica , Peptídeos/química
13.
Bioresour Technol ; 154: 138-47, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24388956

RESUMO

Banana peel (BP), a biomass waste, was converted into a valuable highly porous functional carbon material (HPFCM) by a general chelate-assisted co-assembly process. The HPFCMs were fabricated by using Al(III)-based metal-organic framework-like as a free-standing template and commercial Pluronic F127 as a microstructure-directing agent. Several critical variables for fabrication including doses of Al(III) and F127, carbonization temperature had been optimized and the adsorption behavior of HPFCMs was examined by using methylene blue as dye model compound. The optimal adsorbent was validated as HPFCMs-5-1-800, and its equilibrium data were well fitted to the Langmuir isotherm model with a monolayer adsorption capacity of 385.12 mg g(-1) at ambient temperature. The surface physical properties of HPFCMs-5-1-800 were also exemplarily characterized. The findings revealed that the free-standing template is a potential route for preparation of HPFCM from waste BP.


Assuntos
Biomassa , Carbono/química , Azul de Metileno/isolamento & purificação , Musa/química , Adsorção , Difusão , Concentração de Íons de Hidrogênio , Cinética , Modelos Teóricos , Nitrogênio/química , Porosidade , Soluções , Temperatura Ambiente , Fatores de Tempo
14.
Anal Chem ; 81(24): 10055-60, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19902929

RESUMO

We developed a novel method for rapid screening of carbohydrate-protein interactions using poly(methyl methacrylate) (PMMA) channels statically coated with hydrophobically modified hydroxyethylcellulose (HM-HEC). We found that a self-assembled monolayer (SAM) of HM-HEC on a PMMA surface intact by water allows rapid and reproducible separations of glycan samples using a 20 mM phosphate without HM-HEC. The underlying mechanism for dynamic and static coatings on the PMMA surface is discussed. Simultaneous analysis of the molecular interaction between a complex mixture of carbohydrates from alpha1-acid glycoprotein and proteins has been successfully achieved in PMMA channels statically coated with a SAM of HM-HEC.


Assuntos
Celulose/análogos & derivados , Lectinas/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Plásticos/química , Polissacarídeos/química , Celulose/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Orosomucoide/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polimetil Metacrilato/química , Propriedades de Superfície , Água/química
15.
Langmuir ; 25(16): 9296-301, 2009 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-19459684

RESUMO

Spontaneous adsorption from solution onto solid surface is a common phenomenon in nature, but the force that governs adsorption is still a matter of considerable debate. (1, 2) We found that surfactants and cellulose adsorb from solution onto a poly(methyl methacrylate) (PMMA) surface in an ordered and cooperative way governed by hydrogen bonding. The glucose rings of n-dodecyl-beta-D-maltoside (DDM) and hydroxyethylcellulose (HEC) stand perpendicular to the surface, H-bond to the surface COOMe groups with their C=O and Me-O bonds parallel to the surface, and form a tight monolayer. The non-H-bonded COOMe groups orient their C=O bonds perpendicular to the surface. In contrast, the glucose rings of hydrophobically modified hydroxyethylcellulose (HMHEC) lie flat with the side chains perpendicular to the surface and H-bond to the perpendicular-oriented C=O groups. The non-H-bonded COOMe groups orient their C=O bonds parallel but Me-O bonds near-perpendicular to the surface for stabilizing HMHEC. The current work provides a detailed picture of how surface-active molecules interact with a solid surface and self-assemble into greatly different architectures.


Assuntos
Celulose/química , Interações Hidrofóbicas e Hidrofílicas , Polimetil Metacrilato/química , Tensoativos/química , Adsorção , Sequência de Carboidratos , Celulose/análogos & derivados , Glucosídeos/química , Ligações de Hidrogênio , Microscopia de Força Atômica , Dados de Sequência Molecular , Estrutura Molecular
16.
Biol Pharm Bull ; 29(7): 1487-9, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16819195

RESUMO

For the amplification and ultrafast separation of the genetic markers and DNA sequences that are related to human male infertility, a multiplex PCR for amplifying three DNA sequence-tagged sites (STS) located on the human Y chromosome with possible roles in the spermatogenesis process has been designed and applied followed by separation on a microchip. First, the optimum T(m) degree for the three DNA markers was optimized and determined experimentally, and the three DNA STS were amplified. These three DNA markers were then separated on a 12-lane microchip electrophoresis system, which can analyze the DNA markers on 12 channels simultaneously. The combination of these two technologies, multiplex PCR and microchip electrophoresis, allows the analysis of 36 DNA markers (12x3) within only 180 s.


Assuntos
Infertilidade Masculina/diagnóstico , Infertilidade Masculina/genética , Sequência de Bases , DNA/genética , Primers do DNA , Eletroforese/métodos , Marcadores Genéticos , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
17.
J Chromatogr A ; 1118(2): 218-25, 2006 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-16643931

RESUMO

In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.


Assuntos
Corantes/química , DNA/química , Substâncias Intercalantes/química , Plasmídeos , Células 3T3 , Animais , Núcleo Celular/química , Eletroforese em Microchip , Humanos , Camundongos , Espectrometria de Fluorescência
18.
Anal Chem ; 78(5): 1452-8, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16503593

RESUMO

Hybrid dynamic coating using n-dodecyl beta-d-maltoside (DDM) and methyl cellulose (MC) has been developed for suppression of analyte adsorption and electroosmotic flow (EOF) in a poly(methyl methacrylate) (PMMA) channel. The adsorption of APTS-labeled sugars in a PMMA channel was obviously suppressed with DDM dynamic coating; however, EOF was reduced only by a factor of approximately 25%, resulting in irreproducible separations. In contrast, both analyte adsorption and EOF in a PMMA channel were efficiently minimized with MC coating; however, concentrated MC above 0.3% was required to achieve high-performance separations, which greatly increased viscosity of the solution and caused difficulties during buffer loading and rinsing. In addition, n-dodecyltrimethylammonium chloride did not show observable effects on reducing analyte adsorption, although it has the same hydrophobic alkyl chain as DDM. These results strongly indicated that the polysaccharide moiety of surface modifiers has a specific affinity to surface charges and is crucial to achieving efficient and stable dynamic coating on the PMMA surface. Hybrid dynamic coating with 0.25% DDM and 0.03% MC was found to minimize both analyte adsorption and EOF in a PMMA channel to a negligible level, while still keeping a low viscosity of the solution. High-speed and high-throughput profiling of the N-linked glycans derived from alpha1-acid glycoprotein, fetuin, and ribonuclease B was demonstrated in both single-channel and 10-channel PMMA chips using DDM-MC hybrid coating. We propose that DDM-MC hybrid coating might be a general method for suppressing analyte adsorption and EOF in polymer MCE devices. The current MCE-based method might be a promising alternative for high-throughput screening of carbohydrate alterations in glycoproteins.


Assuntos
Carboidratos/análise , Eletroforese em Microchip/instrumentação , Glucosídeos , Metilcelulose , Técnicas Analíticas Microfluídicas/instrumentação , Polimetil Metacrilato , Adsorção , Eletroforese em Microchip/métodos , Desenho de Equipamento , Glicoproteínas/química , Técnicas Analíticas Microfluídicas/normas , Polissacarídeos/análise
19.
Am J Ind Med ; 49(5): 367-73, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16570259

RESUMO

BACKGROUND: Hexavalent chromium has been extensively investigated regarding its mutagenicity and carcinogenicity; however, its mechanism for initiating and enhancing the development of lung cancer is still obscure. Biomarkers of exposure, effect or susceptibility are required for risk assessment and for epidemiologic research studies especially in occupational settings. Since the surfactant protein system (SP) is very important for normal lung function and for mediating local airway conditions and in the clearance of the upper respiratory tract from the occupational and environmental dusts, we hypothesize that SP genes may represent good candidates to study susceptibility for lung cancer. METHODS: Using PCR genotyping methods with gel electrophoresis and confirmation of results with precise DNA fragment size measurement on microchip electrophoresis, we analyzed SP-B intron-4 polymorphism in 230 subjects who were classified into groups; chromate-related lung cancer, control chromate workers who had not developed lung cancer, control individuals with non chromate-related adenocarcinoma or squamous cell carcinoma of the lungs, or healthy Japanese control individuals. RESULTS: Our results indicated that the SP-B variants (deletion/insertion) were significantly overrepresented (61.3%) in the chromate-related lung cancer group than other groups (X2 = 47.6; DF = 4, P = 0.0001). There was a significant difference between the chromate lung cancer group and both of the control groups, healthy individuals and chromate workers who did not develop lung cancer, showing odds ratios (OR) with 95% confidence intervals (CI) of 21.9 (7.3-65.7) and 19.0 (3.78-95.4), respectively. Compared with 46 non chromate-related SCC of the lung, the SP-B variants were significantly overrepresented in the chromate-related SCC (18/28; 64.3%) than the non-chromate SCC (11/46; 23.9%) of the lung samples (X(2) = 10.27, P = 0.01), OR with 95% CI is 5.73 (2.05-16.01). CONCLUSION: These findings indicate a very strong association of the SP-B intron-4 variants with mechanisms that may enhance lung cancer susceptibility, especially in workers who are employed in chromate industry. Moreover, confirmation of such results may help to suggest adding the SP-B intron-4 typing to be one of the screening tests of the pre-placement medical examination to confirm that the worker has no variations of the SP-B gene before being engaged in a chromium-related industry, with the intention of providing proper medical counseling.


Assuntos
Carcinógenos Ambientais/efeitos adversos , Cromo/efeitos adversos , Neoplasias Pulmonares/genética , Metalurgia , Doenças Profissionais/genética , Precursores de Proteínas/genética , Proteolipídeos/genética , Estudos de Casos e Controles , Intervalos de Confiança , Predisposição Genética para Doença , Genótipo , Humanos , Japão , Neoplasias Pulmonares/induzido quimicamente , Pessoa de Meia-Idade , Exposição Ocupacional/efeitos adversos , Reação em Cadeia da Polimerase
20.
J Chromatogr A ; 1109(2): 138-43, 2006 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-16376899

RESUMO

A novel method for fast profiling of complex oligosaccharides released from glycoproteins based on microchip electrophoresis (mu-CE) is presented here. The characterization of separation conditions, i.e., the composition, concentration and pH of running buffer as well as the applied voltage, has been performed using maltose (G2), cellobiose ( G2'), maltriose (G3) and panose (G3') as oligosaccharide isomer models. In mu-CE, much better separation of oligosaccharide isomers and oligosaccharide ladder was obtained in phosphate buffer than in borate buffer over a wide pH range. Under optimal conditions, high-performance separation of the N-linked complex oligosaccharides released from ribonuclease B, fetuin, alpha1-acid glycoprotein (AGP) and IgG was achieved using polymethylmethacrylate (PMMA) microchips with an effective separation channel of 30 mm. These results represent the first reported analysis of the N-linked oligosaccharides derived from glycoproteins by mu-CE, indicating that the present mu-CE-based method is a promising alternative for characterization of the N-linked oligosaccharides in glycoproteins.


Assuntos
Eletroforese em Microchip/métodos , Glicoproteínas/química , Oligossacarídeos/análise , Sequência de Carboidratos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular
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