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1.
J Mass Spectrom ; 59(5): e5025, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38607249

RESUMO

Energy-resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8 m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo-multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5 µM up to 2.5 µM. The detection limit was 0.08 µM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2 µM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples.


Assuntos
Espectrometria de Massa com Cromatografia Líquida , Espectrometria de Massas em Tandem , Espectrometria de Massas por Ionização por Electrospray , Isótopos , Peptídeos
2.
Anal Bioanal Chem ; 415(29-30): 7211-7221, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37864586

RESUMO

Interferences from isobaric and isomeric compounds represent a common problem in liquid chromatography coupled to mass spectrometry (LC-MS). In this paper, in-source purification and chromatographic separation were combined with the aim of identifying isobaric contamination and quantifying accurately a compound despite the presence of an isobaric co-eluted interference. This is achieved by totally fragmenting in-source the precursor ions of the isobaric interference providing then LC-pseudo-MS2 capability, which allows an accurate quantification without the need for optimizing the chromatographic conditions to separate the co-eluted interference. To illustrate this concept, mixtures of tryptic and non-tryptic peptides were used. The ratio of peak areas of the tryptic peptide and its isotopically labelled internal standard was used not only for quantification with an internal standard calibration curve but also to know (1) if an isobaric interference co-eluted with the tryptic peptide; and (2) what is the minimum cone voltage necessary to ensure the complete removal of isobaric interference. This strategy was applied to quantify the tryptic peptide of two standards with known concentrations and, intentionally contaminated with the isobaric interference. The confidence intervals of the concentrations calculated with the internal standard calibration curve were 8.0 ± 0.5 µM (prepared at 8.0 µM) and 15.7 ± 0.5 µM (prepared at 16.1 µM) that confirm the tryptic peptide can be correctly quantified by in-source purification without the need for improving the chromatographic separation from its isobaric interference.


Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Peptídeos/análise , Calibragem , Padrões de Referência
3.
J Mass Spectrom ; 56(2): e4698, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33480458

RESUMO

The presence of an isobaric contaminant can drastically affect MS and MS/MS patterns leading to erroneous structural and quantitative analysis, which is a real challenge in mass spectrometry. Herein, we demonstrate that MS and MS/MS structural analysis of a compound can be successfully performed despite the presence of an isobaric interference with as low as few millidaltons mass difference by using pseudo-MS3 . To this end, in-source collisional excitation (in-source CID) and the Survival Yield (SY) technique (energy-resolved collision induced dissociation MS/MS) were performed on two different source geometries: a Z-spray and an orthogonal spray (with a transfer capillary) ionization sources on two different mass spectrometers. By using soft ionization conditions, the SY curve for the mixture is a linear combination of the SY curves from the pure compounds demonstrating the presence of two components in the mixture. In the case of harsher ionization conditions, the SY curve of the mixture perfectly overlaps the SY curve from the pure analyte. This observation demonstrates the isobaric interference has been completely removed by in-source CID fragmentation, independently of the source design, leaving then the analyte precursor ions only. Therefore, by measuring the MS spectrum in harsh ionization conditions and according to SY criterium, the compound of interest can be made free from isobaric interference paving the way for, for example, unequivocal HPLC-MS as well as HPLC-MS/MS structural and quantitative analysis despite the presence of a co-eluting isobaric interference.

4.
J Mass Spectrom ; 55(7): e4478, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31834966

RESUMO

Recently, it has been shown that energy-resolved mass spectrometry (MS) can provide quantitative information from two isomeric or isobaric compounds in mixtures by using the survival yield (SY) technique together with "gas-phase collisional purification" (GPCP) strategy (Anal. Chem., 2016, 88, p.10821). Herein, we present an improvement and an extension of this concept to the structural analysis of a model mixture of three isobaric compounds (two peptides and a polyether). By using default collision-induced dissociation (CID) tandem MS parameters on an ion trap instrument, the previous approach did not show any signs of isobaric contamination. However, by modifying CID conditions and using a threefold increase of the He trapping gas pressure (to reach 3.00·10-5 mbar), the SY curve was unexpectedly and strongly shifted to higher excitation voltages with two plateaus appearing. Those plateaus, indicating clearly the presence of three isobaric compounds, were taken as reliable indicators to perform GPCP at carefully selected excitation voltages in order to selectively fragment one compound after the other. In this way, CID mass spectra of each compound were correctly recovered, both in terms of fragment ion peaks and in terms of relative intensities, from energy-resolved MSn spectra of the three compounds mixture. This feature enables their unequivocal structural analysis as if samples were free from isobaric interferences. In this paper, we also discuss the possibility for recovering SY curves for pure compounds directly from the mixture. Clearly, in this case, the higher He trapping gas pressure made it possible to use the SY technique, for the first time, for the structural analysis in the case of mixtures of three isobaric compounds. This observation, quite unexpected, demonstrates that the trapping gas pressure is of paramount importance although it is usually not considered in energy-resolved MS for structural and/or quantitative analysis.

5.
Anal Chem ; 90(24): 14126-14130, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30462486

RESUMO

Model mixtures of isobaric peptides were studied to evaluate the possibility, using tandem mass spectrometry experiments, for internal standard quantification of a tryptic peptide in the presence of an isobaric interference. To this end, direct injection electrospray ionization-tandem mass spectrometry (ESI-MS/MS) experiments were performed on an ion trap instrument using a large mass-selection window (15 m/ z) encompassing the isobaric mixture and the internal standard; MS/MS experiments were carried out to remove completely the interference from the mixture by fragmenting it. This allowed for the correct intensity assignment for the protonated peptide peak and, thus, for the analyte to be quantified through the relative intensity estimate of this peak with respect to the internal standard. This was done by monitoring the 15 m/ z mass-selection window only and without the necessity for careful inspection of any fragment ions peaks. The interference removal was assessed by determining an excitation voltage large enough for the analyte/internal standard ratio to remain constant ensuring correct quantification despite isobaric contamination. A calibration curve was obtained to predict reference samples and compared to reference samples purposely spiked with the interference using the proposed methodology; internal standard quantification of the analyte was made possible with ∼1% deviation despite the isobaric contamination.


Assuntos
Peptídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização por Electrospray/normas , Sequência de Aminoácidos , Calibragem , Gases/química , Peptídeos/síntese química , Peptídeos/normas , Padrões de Referência , Tripsina/metabolismo
6.
Sci Rep ; 8(1): 13038, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158566

RESUMO

Although Ras genes are frequently mutated in human tumors, these mutations are uncommon in breast cancer. However, many breast tumors show evidences of Ras pathway activation. In this manuscript, we have analyzed and characterized mouse mammary tumors generated by random Sleeping Beauty transposon mutagenesis and identify ERAS -a member of the RAS family silenced in adult tissues- as a new gene involved in progression and malignancy of breast cancer. Forced expression of ERAS in human non-transformed mammary gland cells induces a process of epithelial-to-mesenchymal transition and an increase in stem cells markers; these changes are mediated by miR-200c downregulation. ERAS expression in human tumorigenic mammary cells leads to the generation of larger and less differentiated tumors in xenotransplant experiments. Immunohistochemical, RT-qPCR and bioinformatics analysis of human samples show that ERAS is aberrantly expressed in 8-10% of breast tumors and this expression is associated with distant metastasis and reduced metastasis-free survival. In summary, our results reveal that inappropriate activation of ERAS may be important in the development of a subset of breast tumors. These findings open the possibility of new specific treatments for this subset of ERAS-expressing tumors.


Assuntos
Neoplasias da Mama/fisiopatologia , Proteína Oncogênica p21(ras)/metabolismo , Animais , Neoplasias da Mama/patologia , Carcinogênese , Diferenciação Celular , Células Cultivadas , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Neoplasias Experimentais/fisiopatologia , Proteína Oncogênica p21(ras)/genética , Transplante Heterólogo
7.
Anal Bioanal Chem ; 410(23): 5765-5777, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30046869

RESUMO

Intramolecular click-chemistry is increasingly used to generate and control the architecture of complex macromolecules including peptides. Such compounds are, however, very challenging to analyze, in particular quantitatively and also to assess their purity. In this study, tandem mass spectrometry (MS/MS) experiments were carried out with an ion trap mass spectrometer using the Survival Yield (SY) technique to analyze several mixtures of protonated, alkali and alkaline earth metal complexes of two topological linear and cyclic peptide isomers. Univariate (using a single excitation voltage) and multivariate (using several excitation voltages) calibration models have been used. The sensitivity, linearity (R2), intermediate precision (sInt) and error of predicted values (RMSEP) of external calibrations curves have been compared leading to the conclusions that: 1) quantification using tandem mass spectrometry can be performed, with very good performances, for such peptides despite isomerism, 2) quantification is also possible despite the absence of diagnostic fragment ions (possibly independently of the amino-acid sequence), 3) best results are obtained with the largest alkali cation, Cs+, while protonation is highly discouraged, 4) uni/multivariate models show similar performances, but the univariate model may be more suitable for potential applications with direct infusion by electrospray ionization (ESI-MS/MS) and/or matrix-assisted laser desorption ionization (MALDI-MS/MS). Graphical abstract ᅟ.


Assuntos
Química Click/métodos , Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Calibragem , Isomerismo , Análise Multivariada , Peptídeos/síntese química , Peptídeos Cíclicos/análise , Peptídeos Cíclicos/síntese química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
8.
Cancer Res ; 77(6): 1357-1368, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28108518

RESUMO

RAS genes are mutated in 20% of human tumors, but these mutations are very rare in breast cancer. Here, we used a mouse model to generate tumors upon activation of a mutagenic T2Onc2 transposon via expression of a transposase driven by the keratin K5 promoter in a p53+/- background. These animals mainly developed mammary tumors, most of which had transposon insertions in one of two RASGAP genes, neurofibromin1 (Nf1) and RAS p21 protein activator (Rasa1). Immunohistochemical analysis of a collection of human breast tumors confirmed that low expression of RASA1 is frequent in basal (triple-negative) and estrogen receptor negative tumors. Bioinformatic analysis of human breast tumors in The Cancer Genome Atlas database showed that although RASA1 mutations are rare, allelic loss is frequent, particularly in basal tumors (80%) and in association with TP53 mutation. Inactivation of RASA1 in MCF10A cells resulted in the appearance of a malignant phenotype in the context of mutated p53. Our results suggest that alterations in the Ras pathway due to the loss of negative regulators of RAS may be a common event in basal breast cancer. Cancer Res; 77(6); 1357-68. ©2017 AACR.


Assuntos
Biomarcadores Tumorais/metabolismo , Elementos de DNA Transponíveis/genética , Transposases/fisiologia , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/fisiologia , Proteína p120 Ativadora de GTPase/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Estadiamento de Neoplasias , Prognóstico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína p120 Ativadora de GTPase/genética
9.
Anal Chem ; 88(22): 10821-10825, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27805355

RESUMO

The differentiation, characterization, and quantification of isomers and/or isobars in mixtures is a recurrent problem in mass spectrometry and more generally in analytical chemistry. Here we present a new strategy to assess the purity of a compound that is susceptible to be contaminated with another isomeric side-product in trace levels. Providing one of the isomers is available as pure sample, this new strategy allows the detection of isomeric contamination. This is done thanks to a "gas-phase collisional purification" inside an ion trap mass spectrometer paving the way for an improved analysis of at least similar samples. This strategy consists in using collision induced dissociation (CID) multistage mass spectrometry (MS2 and MS3) experiments and the survival yield (SY) technique. It has been successfully applied to mixtures of cyclic poly(L-lactide) (PLA) with increasing amounts of its linear topological isomer. Purification in gas phase of PLA mixtures was established based on SY curves obtained in MS3 mode: all samples gave rise to the same SY curve corresponding then to the pure cyclic component. This new strategy was sensitive enough to detect traces of linear PLA (<3%) in a sample of cyclic PLA that was supposedly pure according to other characterization techniques (1H NMR, MALDI-HRMS, and size-exclusion chromatography). Moreover, in this case, the presence of linear isomer was undetectable according to MS/MS or MS/MS/MS analysis only as fragment ions are also of the same m/z values. This type of approach could easily be implemented in hyphenated mass spectrometric techniques to improve the structural and quantitative analysis of complex samples.

10.
Colloids Surf B Biointerfaces ; 136: 192-200, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26398144

RESUMO

Cationic lipids are used to deliver genetic material to living cells. Their proper biophysical characterization is needed in order to design and control this process. In the present work we characterize some properties of recently synthetized cationic lipophosphoramidates. The studied compounds share the same structure of their hydrophobic backbone, but differ in their hydrophilic cationic headgroup, which is formed by a trimethylammonium, a trimethylarsonium or a dicationic moiety. Dynamic light scattering and cryo-transmission electron microscopy proves that the studied lipophosphoramidates create stable unilamellar vesicles. Fluorescence of polarity probe, Laurdan, analyzed using time-dependent fluorescence shift method (TDFS) and generalized polarization (GP) gives important information about the phase, hydration and dynamics of the lipophosphoramidate bilayers. While all of the compounds produced lipid bilayers that were sufficiently fluid for their potential application in gene therapy, their polarity/hydration and mobility was lower than for the standard cationic lipid - DOTAP. Mixing cationic lipophosphoramidates with DOPC helps to reduce this difference. The structure of the cationic headgroup has an important and complex influence on bilayer hydration and mobility. Both TDFS and GP methods are suitable for the characterization of cationic amphiphiles and can be used for screening of the newly synthesized compounds.


Assuntos
Bicamadas Lipídicas/química , Compostos Organofosforados/química , Cátions , Polarização de Fluorescência , Microscopia Eletrônica de Transmissão/métodos , Água/química
11.
Biophys Chem ; 171: 46-53, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23182464

RESUMO

We studied the physicochemical properties of some cationic lipophosphoramidates used as gene vectors in an attempt to better understand the link between the nature of the hydrophobic chain and both physico-chemical properties and transfection efficiency. These compounds have an arsonium head group and various chain lengths and unsaturation numbers. The synthesis of cationic phospholipids with oleic (Guenin et al., 2000 [1]; Floch et al., 2000 [2]) or linoleic (Fraix et al., 2011 [3]; Le Gall et al., 2010 [4]) chains has already been reported by our group and their efficiency as gene carriers has been demonstrated. Four new compounds were synthesized which incorporated either C14:0, C18:0, C20:4 or C20:5 chains. The membrane fluidity was studied by fluorescence anisotropy measurements. The fusion of liposomes and lipoplexes with membrane models was studied by Förster Resonant Energy Transfer. Finally, DNA condensation was studied and the lipoplexes were tested in vitro to quantify their transfection efficiency. From the results obtained on these cationic lipophosphoramidates series, we show that aliphatic chain length and unsaturation number have an important influence on liposomes physicochemical properties and transfection efficiency. However there is no direct link between fluidity and fusion efficiency or between fluidity and DNA condensation. Nevertheless, it seems that for best transfection efficiency the compounds need to combine the properties of fluidity, fusion efficiency and DNA condensation efficiency. This was the case for the C18:1 and C18:2 compounds.


Assuntos
Amidas/química , Cátions/química , DNA/administração & dosagem , Lipossomos/química , Fosfolipídeos/química , Ácidos Fosfóricos/química , Plasmídeos/administração & dosagem , Amidas/metabolismo , Cátions/metabolismo , Linhagem Celular , DNA/química , DNA/genética , Humanos , Lipossomos/metabolismo , Pulmão/citologia , Fusão de Membrana , Conformação de Ácido Nucleico , Fosfolipídeos/metabolismo , Ácidos Fosfóricos/metabolismo , Plasmídeos/química , Plasmídeos/genética , Transfecção
12.
Anal Bioanal Chem ; 401(9): 3011-7, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21904798

RESUMO

Classical photo-induced fluorescence methods are conducted in two steps: a UV irradiation step in order to form a photo-induced compound followed by its fluorimetric determination. Automated flow injection methods are frequently used for these analyses. In this work, we propose a new method of direct laser photo-induced fluorescence analysis. This new method is based on direct irradiation of the analyte in a fluorimetric cell in order to form a photo-induced fluorescent compound and its direct fluorimetric detection during a short irradiation time. Irradiation is performed with a tuneable Nd:YAG laser to select the optimal excitation wavelength and to improve the specificity. It has been applied to the determination of bisphenol A, an endocrine disrupter compound that may be a potential contaminant for food. Irradiation of bisphenol A at 230 nm produces a photo-induced compound with a much higher fluorescence quantum yield and specific excitation/emission wavelengths. In tap water, the fluorescence of bisphenol A increases linearly versus its concentration and, its determination by direct laser photo-induced fluorescence permits to obtain a low limit of detection of 17 µg L(-1).

13.
Anal Bioanal Chem ; 399(1): 119-26, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20632162

RESUMO

In the present study, we have used carbon nanotube field effect transistors (FET) that have been functionalized with protein G and IgG to detect Aspergillus flavus in contaminated milled rice. The adsorbed protein G on the carbon nanotubes walls enables the IgG anti-Aspergillus antibodies to be well oriented and therefore to display full antigen binding capacity for fungal antigens. A solution of Tween 20 and gelatine was used as an effective blocking agent to prevent the non-specific binding of the antibodies and other moulds and also to protect the transducer against the interferences present in the rice samples. Our FET devices were able to detect at least 10 µg/g of A. flavus in only 30 min. To evaluate the selectivity of our biosensors, Fusarium oxysporum and Penicillium chrysogenum were tested as potential competing moulds for A. flavus. We have proved that our devices are highly selective tools for detecting mycotoxigenic moulds at low concentrations in real samples.


Assuntos
Aspergillus flavus/química , Técnicas Biossensoriais/métodos , Contaminação de Alimentos/análise , Nanotubos de Carbono/química , Oryza/microbiologia , Aspergillus flavus/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Transistores Eletrônicos
14.
Methods Mol Biol ; 625: 213-25, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20422393

RESUMO

We describe in detail the different steps involved in the construction of a carbon nanotube field-effect transistor (CNTFET) based on a network of single-walled carbon nanotubes (SWCNTs), which can selectively detect human immunoglobulin G (HIgG). HIgG antibodies, which are strongly adsorbed onto the walls of the SWCNTs, are the basic elements of the recognition layer. The nonspecific binding of proteins or other interferences are avoided by covering the nonadsorbed areas of the SWCNTs with Tween 20. The CNTFET is a reagentless device that does not need labels to detect HIgG.


Assuntos
Anticorpos/análise , Técnicas Biossensoriais , Imunoglobulina G/análise , Nanotubos de Carbono/química , Transistores Eletrônicos , Adsorção , Eletrodos , Humanos , Polissorbatos/química , Propriedades de Superfície
15.
Analyst ; 135(5): 1037-41, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20419254

RESUMO

A facile, solid-contact selective potentiometric aptasensor exploiting a network of single-walled carbon nanotubes (SWCNT) acting as a transducing element is described in this work. The molecular properties of the SWCNT surface have been modified by covalently linking aptamers as biorecognition elements to the carboxylic groups of the SWCNT walls. As a model system to demonstrate the generic application of the approach, a 15-mer thrombin aptamer interacts with thrombin and the affinity interaction gives rise to a direct potentiometric signal that can be easily recorded within 15 s. The dynamic linear range, with a sensitivity of 8.0 mV/log a(Thr) corresponds to the 10(-7)-10(-6) M range of thrombin concentrations, with a limit of detection of 80 nM. The aptasensor displays selectivity against elastase and bovine serum albumin and is easily regenerated by immersion in 2 M NaCl. The aptasensor demonstrates the capacity of direct detection of the recognition event avoiding the use of labels, mediators, or the addition of further reagents or analyte accumulation.


Assuntos
Aptâmeros de Nucleotídeos/química , Nanotubos de Carbono/química , Potenciometria/métodos , Trombina/análise , Técnicas Biossensoriais , Humanos , Sensibilidade e Especificidade
16.
Biosens Bioelectron ; 25(2): 344-9, 2009 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-19656669

RESUMO

A new solid-contact ion-selective electrode has been developed for determining choline and derivatives in aqueous solutions. The backbone of this new potentiometric sensor is the conjunction of the cavitand receptor, as the molecular recognition element, and a network of non-carboxylated single-walled carbon nanotubes, acting as a solid transducer material. The octaamide cavitand, a synthetic receptor that is highly selective for biologically important trimethyl alkylammonium cations such as choline, acetylcholine or carnitine, makes the selective determination of these compounds possible for the first time. The guest-host interaction takes place in the acrylate ion-selective membrane of the solid-contact electrode. The sensor was characterized by electrochemical impedance spectroscopy and environmental scanning electron microscopy. The new electrode displays a nearly Nernstian slope (57.3+/-1.0 mV/decade) and very stable behaviour (DeltaE/Deltat=224 muVh(-1)) throughout the dynamic range (10(-5) to 10(-1)M). The limit of detection of 10(-6.4)M and the high selectivities obtained will enable choline and derivatives to be determined in biological samples. Finally, the stability of the electrical potential of the new solid-contact electrode was examined by performing current-reversal chronopotentiometry and the influence of the interfacial water film was evaluated by the potentiometric water layer test.


Assuntos
Amidas/química , Técnicas Biossensoriais/instrumentação , Colina/análise , Eletroquímica/instrumentação , Eletrodos , Éteres Cíclicos/química , Nanotubos de Carbono/química , Resorcinóis/química , Íons , Nanotecnologia/instrumentação , Nanotubos de Carbono/ultraestrutura , Transdutores
17.
Diagn Cytopathol ; 37(9): 647-53, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19306424

RESUMO

Single primer pair polymerase chain reaction (PCR) assays for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements and immunophenotyping by flow cytometry have been proved as useful techniques in the diagnosis of lymphoid disorders in fine needle aspirates. However, a comparative analysis of both ancillary techniques in the same samples has not been previously performed. To compare the sensitivity of flow cytometry and PCR techniques, we made a wide prospective study of 77 fine needle aspiration biopsy (FNAB) samples from lymph nodes and extranodal lymphoid infiltrates. The adjunctive values of a single primer pair PCR amplification of IgH genes and of the immunophenotyping by flow cytometry were evaluated comparing their results with the final clinicopathological diagnosis of each patient supported by histological features and clinical follow up. Among the 24 B-cell non-Hodgkin lymphomas, monoclonal IgH bands were detected in 22 cases by PCR, and 21 cases were correctly considered B-cell lymphoma by flow cytometry. A monoclonal IgH band was also detected in 1 of the 53 reactive lymphoid disorders. When both ancillary techniques were combined with morphological findings, 23 of the 24 B-cell lymphomas were correctly diagnosed but one reactive lymphoid disorder was also considered a B-cell lymphoma. We demonstrate a similar level of detection of B-cell lymphomas by single round PCR and flow cytometry techniques, and a strong adjunctive value when combined with morphological findings to diagnose correctly lymphoproliferative disorders by FNAB. However, we must be cautious with PCR results since false-positive cases can occur.


Assuntos
Citometria de Fluxo , Linfoma de Células B/diagnóstico , Reação em Cadeia da Polimerase , Biópsia por Agulha Fina , Diagnóstico Diferencial , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunofenotipagem , Linfoma de Células B/genética , Sensibilidade e Especificidade
18.
Analyst ; 133(8): 1001-4, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18645639

RESUMO

We report a new type of ion-sensitive field effect transistor (ISFET). This type of ISFET incorporates a new architecture, containing a network of single-walled carbon nanotubes (SWCNTs) as the transduction layer, making an external reference electrode unnecessary. To show an example of its application, the SWCNT-based ISFET is able to detect at least 10(-8) M of potassium in water using an ion-selective membrane containing valinomycin.

19.
Analyst ; 133(8): 1005-8, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18645640

RESUMO

We report a field effect transistor (FET) based on a network of single-walled carbon nanotubes (SWCNTs) which can selectively detect human immunoglobulin G (HIgG). HIgG antibodies, which are strongly adsorbed onto the walls of the SWCNTs, are the basic elements of the recognition layer. The non-specific binding of proteins and the effects of other interferences are avoided by covering the non-adsorbed areas of the SWCNTs with Tween 20. The selectivity of the sensor has been tested against bovine serum albumin (BSA), the most abundant protein in plasma. HIgG in aqueous solution with concentrations from 1.25 mg L(-1) (8 nM) can be readily detected with response times of about 10 min. The SWCNT networks that form the basis of the sensor are easily grown by chemical vapour deposition. Silver screen-printed electrodes make the sensor quick to build. The sensitivity obtained with this sensor is similar to other FET devices based on SWCNTs built using much more complicated lithography processes. Moreover, the sensor is a reagentless device that does not need labels to detect HIgG.


Assuntos
Imunoglobulina G/sangue , Eletroquímica/instrumentação , Eletroquímica/métodos , Eletrodos , Humanos , Nanotubos de Carbono , Prata , Transistores Eletrônicos
20.
Biosens Bioelectron ; 24(2): 279-83, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18495470

RESUMO

In this paper we report a fast, sensitive and label-free biosensor for the selective determination of Salmonella Infantis. It is based on a field effect transistor (FET) in which a network of single-walled carbon nantotubes (SWCNTs) acts as the conductor channel. Anti-Salmonella antibodies were adsorbed onto the SWCNTs and subsequently the SWCNTs were protected with Tween 20 to prevent the non-specific binding of other bacteria or proteins. Our FET devices were exposed to increasing concentrations of S. Infantis and were able to detect at least 100 cfu/mL in 1h. To evaluate the selectivity of our FET devices, Streptococcus pyogenes and Shigella sonnei were tested as potential competing bacteria for Salmonella. At a concentration of 500 cfu/mL, neither Streptococcus nor Shigella interfered with the detection of Salmonella. Therefore, these devices could be used as useful label-free platforms to detect S. Infantis and, by using the suitable antibody, other bacteria or viruses.


Assuntos
Técnicas Biossensoriais/instrumentação , Contagem de Colônia Microbiana/instrumentação , Microeletrodos , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestrutura , Salmonella/isolamento & purificação , Transistores Eletrônicos , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Propriedades de Superfície
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