RESUMO
Bloodstream bacterial infections are life-threatening conditions necessitating prompt medical care. Rapid pathogen identification is essential for early setting of the best anti-infectious therapy. However, the bacterial load in blood samples from patients with bacteremia is too low and under the limit of detection of most methods for direct identification of bacteria. Therefore, a preliminary step enabling the bacterial multiplication is required. To do so, blood cultures still remain the gold standard before bacteremia diagnosis. Bacterial identification is then usually obtained within 24 to 48 hours -at least- after blood sampling. In the present work, the fast and direct identification of bacteria present in blood cultures is completed in less than 12 hours, during bacterial growth, using an antibody microarray coupled to a Surface Plasmon Resonance imager (SPRi). Less than one bacterium (Salmonella enterica serovar Enteritidis) per milliliter of blood sample is successfully detected and identified in blood volumes similar to blood tests collected in clinics (i.e. several milliliters). This proof of concept demonstrates the workability of our method for human samples, despite the highly complex intrinsic nature of unprocessed blood. Our label-free method then opens new perspectives for direct and faster bacterial identification in a larger range of clinical samples.
Assuntos
Bacteriemia/diagnóstico , Hemocultura/métodos , Infecções por Salmonella/diagnóstico , Salmonella enteritidis/fisiologia , Testes Sorológicos/métodos , Anticorpos Antibacterianos/metabolismo , Testes Diagnósticos de Rotina , Diagnóstico Precoce , Estudos de Viabilidade , Testes Hematológicos , Humanos , Análise Serial de Proteínas , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
UNLABELLED: Among bacterial pathogens involved in food-illnesses, seven serogroups (O26, O45, O103, O111, O121, O145 and O157) of Shiga-toxin producing Escherichia coli (STEC), are frequently identified. During such outbreak, and due to the perishable property of most foodstuff, the time laps for the identification of contaminated products and pathogens is thus critical to better circumvent their spread. Traditional detection methods using PCR or culture plating are time consuming and may present some limitations. In this study, we present a multiplexed immunoassay for the optical detection of most commonly enterohemorrhagic E. coli serogroups: O26, O45, O103, O111, O121, O145 and O157:H7 in a single device. The use of Surface Plasmon Resonance imaging not only enabled the label-free analysis of the samples but gave results in a real-time manner. A dedicated protocol was set up for the detection of both low contaminating bacterial concentrations of food samples (5 CFU per 25 g) and postenrichment aliquots. By combining one single device for the detection of O157 and non-O157 STEC in a label-free manner, this rapid approach may have an important economic and societal impact. SIGNIFICANCE AND IMPACT OF THE STUDY: This article presents a simple-to-operate immunoassay for the specific detection of Shiga-toxin producing Escherichia coli (STEC). This approach consists in the on-chip assay detection of viable cells on a specifically designed antibody microarray. By skipping any enrichment step and avoiding the use of labelling agent, this approach based on the Surface Plasmon Resonance imaging of the microarrays turns out to be much faster and more cost effective by comparison with standardized methods.
Assuntos
Imunoensaio/métodos , Tipagem Molecular/métodos , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/genética , Ressonância de Plasmônio de Superfície/métodos , Proteínas de Escherichia coli/genética , Doenças Transmitidas por Alimentos/microbiologia , Reação em Cadeia da PolimeraseRESUMO
AIMS: Rapid detection of enterohaemorrhagic E. coli O157:H7 in large range of stress conditions occurring in food processing. METHODS AND RESULTS: Detection of E. coli O157:H7 in various food processing stress conditions using surface plasmon resonance imaging (SPRi) technique on an antibody microarray was evaluated. The direct detection method based on the culture/capture/measure (CCM) process consists of detecting bacteria during an enrichment step, which significantly decreases the overall assay duration. In optimized culture conditions, this method allows the specific detection of low CFU ml(-1) in <7 h. Detection of bacteria directly in contaminated food samples was also conducted. CONCLUSIONS: The CCM technique using an antibody microarray is a label-free immunoassay that allows rapid detection of E. coli O157:H7 in both food processing stress conditions and complex food matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is promising for detecting E. coli O157:H7 at different steps of food and drink processing and during storage. SPRi appears to be a suitable and powerful detection method for routine quality controls in food industry with important economic and societal impact.
Assuntos
Escherichia coli O157/isolamento & purificação , Microbiologia de Alimentos/métodos , Ressonância de Plasmônio de Superfície/métodos , Manipulação de Alimentos , Imunoensaio/métodos , Carne/microbiologia , Estresse FisiológicoRESUMO
Conditions for simple derivatization of reducing carbohydrates via adipic acid dihydrazide microwave-assisted condensation are described. We demonstrate with a diverse set of oligo- and polysaccharides how to improve a restrictive and labor intensive conventional conjugation protocol by using microwave-assisted chemistry. We show that 5 min of microwave heating in basic or acidic conditions are adequate to generate, in increased yields, intact and functional glycosylhydrazides, whereas hours to days and acidic conditions are generally required under conventional methods.
Assuntos
Carboidratos/síntese química , Micro-Ondas , Sequência de Carboidratos , Carboidratos/química , Ressonância de Plasmônio de SuperfícieRESUMO
DNA microarrays find applications in an increasing number of domains where more quantitative results are required. DNA being a charged polymer, the repulsive interactions between the surface of the microarray and the targets in solution are increasing upon hybridization. Such electrostatic penalty is generally reduced by increasing the salt concentration. In this article, we present equilibrium-melting curves obtained from dedicated physicochemical experiments on DNA microarrays in order to get a better understanding of the electrostatic penalty incurred during the hybridization reaction at the surface. Various salt concentrations have been considered and deviations from the commonly used Langmuir adsorption model are experimentally quantified for the first time in agreement with theoretical predictions.
Assuntos
DNA/química , Análise de Sequência com Séries de Oligonucleotídeos , Sais/farmacologia , Sequência de Bases , Fenômenos Químicos/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA/genética , Relação Dose-Resposta a Droga , Ouro/química , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Hibridização de Ácido Nucleico/efeitos dos fármacos , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/genética , Polimerização/efeitos dos fármacos , Pirróis/química , Eletricidade Estática , Enxofre/química , TermodinâmicaRESUMO
An original oligonucleotide-array, coupled with SPR-imaging detection, has been developed to study biological interactions between DNA base lesions and DNA repair enzymes. This bioanalytical tool constitutes an efficient screening platform to quantify DNA repair activities and to search for new DNA repair inhibitors.
Assuntos
Dano ao DNA , Reparo do DNA , DNA-Formamidopirimidina Glicosilase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ressonância de Plasmônio de Superfície , 8-Hidroxi-2'-Desoxiguanosina , Desoxiadenosinas/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Proteínas de Escherichia coli/metabolismoRESUMO
The detection of point mutations in genes presents clear biological and medical interest. Various methods have been considered. In this paper, we take advantage of surface plasmon resonance imaging, a technique allowing detection of unlabeled DNA hybridization. Coupled with a temperature scan, this approach allows us to determine the presence of single-point mutations in oligonucleotide samples from the analysis of DNA's melting curves in either the homozygous or heterozygous case. Moreover, these experimental data are confirmed in good agreement with numerical calculations.
Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Hibridização In Situ/métodos , Mutação Puntual , Ressonância de Plasmônio de Superfície/métodos , Sequência de Bases , Modelos Biológicos , Sensibilidade e Especificidade , Temperatura , Fatores de TempoRESUMO
We present an analysis of hybridization experiments on a DNA chip studied by surface plasmon resonance imaging. The reaction constants at various temperatures and for different probe lengths are obtained from Langmuir isotherms and hybridization kinetics. The melting curves from temperature scans are also obtained without any labeling of the targets. The effects of the probe length on the hybridization thermodynamics, deduced from the temperature dependence of the reaction constants as well as from the melting curves, suggest dispersion in the length of the hybridization segments of the probes accessible to the targets. Those are, however, sufficient to suggest efficient point mutation detection from temperature scans.
Assuntos
Artefatos , Análise Mutacional de DNA/métodos , DNA/química , DNA/genética , Hibridização In Situ/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Ressonância de Plasmônio de Superfície/métodos , DNA/análise , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Transição de Fase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Temperatura , Temperatura de TransiçãoRESUMO
In the field of DNA sensing, DNA hybridisation detection is generally performed by fluorescence microscopy. However, fluorescence instrumentation is difficult to miniaturise in order to produce fully integrated DNA chips. In this context, electrochemical detection of DNA hybridisation may avoid this limitation. Therefore, the use of DNA intercalators is particularly attractive due to their selectivity toward DNA double strand enabling DNA labelling without target chemical modification and, for most of them, to their electroactivity. We have synthesized a pyridoacridone derivative dedicated to DNA hybridisation electrochemical-sensing which presents good electrochemical reversibility, electroactivity at mild potentials and specificity toward DNA double strand. The electrochemical behaviour of this molecule has been assessed using cyclic voltammetry (CV). DNA/intercalator interactions were studied by differential pulse voltammetry (DPV) before application to hybridisation detection onto DNA sensors based on polypyrrole modified electrodes.
Assuntos
Acridinas/análise , Acridinas/química , Técnicas Biossensoriais/métodos , DNA/análise , DNA/química , Eletroquímica/métodos , Hibridização de Ácido Nucleico/métodos , Acridonas , Técnicas Biossensoriais/instrumentação , Hibridização in Situ Fluorescente/métodos , Substâncias Intercalantes/análise , Substâncias Intercalantes/química , Oxirredução , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem/métodosRESUMO
Biotinylated bacteria were immobilized onto biotin/avidin modified electrode surfaces. Firstly, an electrospotting deposition method, followed by fluorescence microscopy, showed that bacteria were specifically grafted onto a gold surface. Fluorescence intensity versus the quantity of bacteria deposited on the surface was correlated, allowing determination of the microbial saturation point. Secondly, biotinylated bacteria were immobilized onto a glassy carbon macro-electrode in order to assess immobilized bacterial denitrification activity. During a 7-day trial, the modified electrode completely denitrified 5 mM nitrate, with a rate of 1.66 mM/day over the first 3 days. When the same electrode was placed in fresh nitrate solution, the denitrification rate dropped to 0.80 mM/day. Crucially, the immobilized bacteria did not become detached from the electrode during the study.
Assuntos
Aderência Bacteriana/fisiologia , Técnicas Biossensoriais/métodos , Técnicas de Cultura de Células/métodos , Eletroquímica/métodos , Eletrodos , Paracoccus pantotrophus/citologia , Polímeros/química , Pirróis/química , Bioensaio/métodos , Biotinilação/métodos , Sobrevivência Celular/fisiologia , Células Imobilizadas/fisiologia , Materiais Revestidos Biocompatíveis/química , Indicadores e Reagentes , Nitratos/metabolismo , Paracoccus pantotrophus/metabolismoRESUMO
BACKGROUND: Detection of mutations in cancer-related genes is of major importance for both basic knowledge and clinical practice. Several strategies have been developed to diagnose these alterations. We describe a method based on polypyrrole DNA chip technology to detect K-ras gene mutations in tumors. METHODS: An oligodeoxynucleotide array was constructed on a silicon device by copolymerization of 5'-pyrrole-labeled oligodeoxynucleotides and pyrrole. The samples to be analyzed were then amplified by PCR, and the single-stranded biotin-labeled amplified DNA was specifically hybridized to the addressed probes. Perfectly matched duplexes were detected by fluorescence microscopy using R-phycoerythrin as the detection label. The developed methodology was applied to genotype assignment of K-ras in human samples. The genotypes of 75 DNA genomic samples from colorectal cancer patients were analyzed side by side using direct DNA sequencing and a polypyrrole DNA chip. RESULTS: The chip method unequivocally defined all of the genotypes. Mutations present at <10% of the wild-type DNA concentration could be distinguished. CONCLUSIONS: This probe array assay is a rapid and reliable procedure that may be used to detect mutations.
Assuntos
Neoplasias Colorretais/genética , DNA de Neoplasias/genética , Genes ras , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Oncogênica p21(ras)/genética , Polímeros , Pirróis , Genótipo , Humanos , Microscopia de Fluorescência , Mutação , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
Conducting polymer films, such as polypyrrole, appear particularly attractive for the immobilisation of biological molecules by entrapment or covalent grafting. We describe here a new pyrrole phosphorarnidite building block allowing the synthesis of oligonucleotide (ODN) bearing a pyrrole moiety. The electropolymerisable pyrrole moiety was then introduced on the 5' end of the oligonucleotide. The electrosynthesis of a copolymer, from solutions containing pyrrole and pyrrole-ODN, gives in one step strongly adhesive films containing ODN probes at electrode surfaces. In this contribution, we have used such a methodology to verify its feasibility for the modification of quartz crystal microbalance (QCM) electrodes. The obtained biosensors enable the detection of DNA hybridisation in real time by micro-gravimetric transduction. Finally, as DNA targets were previously modified by biotin, we have used the affinity between biotin and avidin to validate the effectiveness of QCM transduction by fluorescence microscopy and to amplify the recorded micro-gravimetric signal.
RESUMO
We describe in this paper a methodology to quantify multispot parallel DNA hybridizations and denaturations on gold surfaces by using, on one hand, a polypyrrole-based surface functionalization based on an electrospotting process and, on the other hand, surface plasmon resonance imaging allowing real-time measurements on several DNA spots at a time. Two characterization steps were performed in order to optimize the immobilization of oligonucleotide probes and, thus, to increase the signal-to-noise ratio of monitored hybridization signals: the first step consisted of characterizing the signal dependence upon the density of immobilized 15-mer probes, and, the second step, in analyzing the hybridization response versus spot thickness. We further demonstrated that a surface density of polypyrrole/DNA probes of approximately 130 fmol/ mm2 (590 pg/mm2) optimizes the hybridization signal that can be detected directly. Optimal thickness of the spot was found to be close to 11 nm. Specificity and regeneration steps on each spot have also been demonstrated successfully, showing this method to be very competitive and convenient in use.
Assuntos
DNA/química , Polímeros/química , Pirróis/química , Sequência de Bases , Sondas de DNA , Hibridização de Ácido Nucleico , Ressonância de Plasmônio de SuperfícieRESUMO
Biosensors based on electronic conducting polymers appear particularly well suited to the requirements of modern biological analysis--multi-parametric assays, high information density, and miniaturization. We describe a new methodology for the preparation of addressed DNA matrices. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5' end a pyrrole moiety. The resulting polymer film deposited on the addressed electrode consists of pyrrole chains bearing covalently linked oligonucleotides (ODN). An oligonucleotide array was constructed on a silicon device bearing a matrix of 48 addressable 50 x 50 microns gold microelectrodes. This technology was successfully applied to the genotyping of hepatitis C virus in blood samples. Fluorescence detection results show good sensitivity and a high degree of spatial resolution. In addition, gravimetric studies carried out by the quartz crystal microbalance technique provide quantitative data on the amount of surface-immobilized species. In the case of ODN, it allows discrimination between hybridization and nonspecific adsorption. The need for versatile processes for the immobilization of biological species on surfaces led us to extend our methodology. A biotinylated surface was obtained by coelectropolymerization of pyrrole and biotin-pyrrole monomers. The efficiency for recognition (and consequently immobilization) of R-phycoerythrin-avidin was demonstrated by fluorescence detection. Copolymerization of decreasing ratios of pyrrole-biotin over pyrrole allowed us to obtain a decreasing scale of fluorescence.
Assuntos
Técnicas Biossensoriais/instrumentação , Eletroquímica/instrumentação , Eletroquímica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Polímeros/química , Adsorção , Biotina/química , Biotinilação , Eletrodos , Genótipo , Ouro/química , Hepacivirus/genética , Modelos Químicos , Oligonucleotídeos/química , Pirróis/química , Quartzo , Silício/química , Fatores de TempoRESUMO
We wish to show in this paper new developments and new applications of the pyrrole copolymerization process allowing the addressing of pyrrole-modified biomolecules on microelectrode arrays. Two main developments are described: the first one concerns the development of multiplexed silicon chips bearing 128 microelectrodes instead of 48 for the passive chips. The second one deals with new applications of this grafting process concerning not only DNA chips but peptide chips too. In this way, copolymerization of pyrrole peptides on the chip (leading to peptide chip) and their immunological detection is illustrated. This technology shows a high dimensional resolution and a real versatility.
Assuntos
DNA/análise , Microeletrodos , Peptídeos/análise , Polímeros , Pirróis , Silício , Animais , DNA/química , Humanos , Peptídeos/químicaRESUMO
We describe in this article an oligonucleotide array constructed on a silicon device bearing a matrix of addressable 50-microns microelectrodes. Each electrode was covered by a conducting polymer (polypyrrole) grafted by an oligonucleotide (ODN). The DNA chip was prepared by successive electrochemically addressed copolymerizations of 5' pyrrole-labeled ODN and pyrrole. Following hybridization of the biotinylated amplified sample on the chip bearing a series of probes, detection was carried out by fluorescence microscopy through an R-phycoerythrin label. This technology was successfully applied to the genotyping of hepatitis C virus in blood samples. Results show good sensitivity and a high degree of dimensional resolution.
Assuntos
Biotecnologia/instrumentação , DNA Viral/análise , DNA/química , Hepacivirus/genética , Pirróis/química , Silício , DNA/genética , Eletroquímica , Genótipo , Microeletrodos , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Controle de Qualidade , Sensibilidade e EspecificidadeRESUMO
In the field of biological analysis, the need for multiparametric analysis has prompted the development of supports bearing a series of biomolecules linked to a support in a precise location (addressed). To reach a high information density, miniaturization of this kind of support has to be carried out. We describe in this paper an approach involving the use of electro-conducting polymers such as polypyrrole. This technology is based on an electro-directed copolymerization of pyrrole and oligodeoxynucleotides (ODN) linked to a pyrrole residue. The process allows the grafting of the selected ODN at the surface of the successively addressed microelectrodes. In this way, the syntheses are carried out on 50 microm electrodes on passive chips or on active (multiplexed) chips bearing 48 or 128 gold microelectrodes, respectively. The detection of biological targets recognized by the biochip is carried out by using fluorescent tracers. This technology, involving prepurified materials precisely addressed, allows better reproducibility of the biochip preparation and, then, an easy interpretation of the fluorescence results. The versatility of this technology is illustrated by ODN or peptide copolymerizations leading to DNA chips or peptide chips, respectively. This would open the field for other biological interaction studies.
Assuntos
Microeletrodos , Polímeros , Imunoensaio , Microscopia de Fluorescência , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Pirróis/química , Reprodutibilidade dos TestesRESUMO
A new methodology for the preparation of addressed DNA matrices is described. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5' end a pyrrole moiety introduced by phosphoramidite chemistry. The electro-controlled synthesis of the copolymer (poly-pyrrole) gives, in one step, a solid conducting film deposited on the surface of an electrode. The resulting polymer consists of pyrrole chains bearing covalently linked oligonucleotide. The polymer growth is limited to the electrode surface, so that it is possible to prepare a DNA matrix on a multiple electrode device by successive copolymerizations. A support bearing four oligonucleotides was used to detect three ras mutations on a synthetic DNA fragment.
Assuntos
DNA/análise , Oligonucleotídeos/síntese química , Polímeros/química , Pirróis/síntese química , Amidas/síntese química , Sequência de Bases , Eletroquímica , Eletrodos , Genes ras , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , Ácidos Fosfóricos/síntese químicaRESUMO
A nonisotopic homogeneous detection of nucleic acid sequences after amplification is described. We show that a DNA fragment bearing T7 RNA polymerase promoters on each extremity is able to be transcribed in two complementary RNAs, leading to a high yield direct synthesis of double-stranded RNA (dsRNA). Thus, this dsRNA can be easily detected and quantified in solution by fluorescence in the presence of propidium iodide. This reaction, used as a postamplification step, has been associated with a nested polymerase chain reaction (PCR); the second PCR round allowing the incorporation of the T7 promoters. This leads to a very efficient homogeneous assay. The fluorescence signal is proportional to the concentration of PCR product and is highly specific. This method can be easily carried out with currently available reagents and with unsophisticated instrumentation. This homogeneous procedure has been evaluated for the detection of HIV1 in blood samples; the sensitivity and the specificity appear to be equivalent to that of the radioactive method.
Assuntos
DNA Viral/análise , DNA Viral/genética , HIV-1/genética , RNA de Cadeia Dupla/análise , Sequência de Bases , Primers do DNA , DNA Viral/sangue , RNA Polimerases Dirigidas por DNA/genética , Fluorescência , Fluorometria/métodos , Amplificação de Genes , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/genética , Sensibilidade e Especificidade , Transcrição Gênica , Proteínas ViraisRESUMO
A quick and sensitive dot-blot assay using non-radioactive labelled RNA probes was developed for the detection of the CF53 strain of hepatitis A virus (HAV) in cell culture. The cDNA of the 5' end of the HM175 strain was inserted in a transcription vector pSPT18 and was used to synthesize 32P- or digoxigenin-labelled RNA probes. These RNA probes specifically detected the RNA of the CF53 strain and can be used to detect HAV in PLC/PRF/5 cells. The sensitivity of non-radioactive tests was comparable to that of radiolabelled probes.