RESUMEN
A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.
Asunto(s)
Antígenos Bacterianos/análisis , Enterotoxinas/análisis , Análisis por Matrices de Proteínas , Staphylococcus aureus/química , Staphylococcus epidermidis/inmunología , Factores de Virulencia/análisis , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/inmunología , Anticuerpos Inmovilizados , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Técnicas Biosensibles , Electroquímica/métodos , Enterotoxinas/inmunología , Enterotoxinas/orina , Contaminación de Alimentos , Microbiología de Alimentos , Leche/química , Factores de Virulencia/inmunología , Factores de Virulencia/orinaRESUMEN
Microarrays have become an increasingly important tool for biotechnology and molecular diagnostics. Despite many advantages, their sensitivity is still insufficient for such tasks as the analysis of small sample quantities and for the detection of alterations in gene expression of low-abundance genes. Accordingly, amplification strategies are necessary. Approaches to amplify the signal intensity include the increase of the number of dye molecules per target through either particle labels or rolling circle amplification, as used for this study.
Asunto(s)
ADN Circular/química , ADN/química , Fluorescencia , Colorantes Fluorescentes/farmacología , Oligonucleótidos/química , Carbocianinas/química , ADN Ligasas , ADN de Cadena Simple , Modelos Químicos , Modelos Genéticos , Técnicas de Amplificación de Ácido Nucleico , Conformación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Sondas de Oligonucleótidos/químicaRESUMEN
Sensitive detection assays are a prerequisite for the analysis of small amounts of samples derived from biological material. There is a great demand for highly sensitive and robust detection techniques to analyze biomolecules. The combination of catalytic active DNA (DNAzyme) with a peroxidase activity with rolling circle amplification (RCA) is a promising alternative to common detection systems. The rolling circle amplification leads to a product with tandemly linked copies of DNAzymes. The continuous signal generation of the amplified DNAzymes results in an increased sensitivity. The combination of two amplification reactions, namely RCA and DNAzymes, results in increased signal intensity by a factor of 10(6). With this approach the labeling of samples can be avoided. The advantage of the introduced assay is the usage of nucleic acids as biosensors for the detection of biomolecules. Coupling of the analyte molecule to the detection molecules allows the direct detection of the analyte molecule. The described label-free hotpot assay has a broad potential field of applications. The hotpot assay can be adapted to detect and analyze RNA, DNA and proteins down to femtomolar concentrations in a miniaturized platform with a total reaction solution of 50 nl. The applicability of the assay for diagnostics and research will be shown with a focus on high throughput systems using a nano-well platform.