RESUMEN
An electrochemical biosensor has been developed for detection of Escherichia coli O157 by integrating lateral flow with screen-printed electrodes. The screen-printed electrodes were attached under the lateral flow detection line, and organic-inorganic nanoflowers prepared from E. coli O157-specific antibodies as an organic component were attached to the lateral flow detection line. In the presence of E. coli O157, an organic-inorganic nanoflower-E. coli O157-antimicrobial peptide-labelled ferrocene sandwich structure is formed on the lateral flow detection line. Differential pulse voltammetry is applied using a smartphone-based device to monitor ferrocene on the detection line. The resulting electrochemical biosensor could specifically detect E. coli O157 with a limit of detection of 25 colony-forming units mL-1. Through substitution of antibodies of organic components in organic-inorganic nanoflowers, biosensors have great potential for the detection of other pathogens in biomedical research and clinical diagnosis.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Escherichia coli O157 , Escherichia coli O157/aislamiento & purificación , Escherichia coli O157/inmunología , Técnicas Biosensibles/métodos , Inmunoensayo/métodos , Inmunoensayo/instrumentación , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Límite de Detección , Nanoestructuras/química , Electrodos , Compuestos Ferrosos/química , Anticuerpos Inmovilizados/inmunología , Metalocenos/química , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/inmunología , Péptidos Antimicrobianos/químicaRESUMEN
To prevent foodborne diseases and minimize their impacts, it is extremely important to develop a cost-effective and efficient bacterial detection assay for diagnostics, particularly in resource-poor settings. In this study, 16S rRNA from foodborne Salmonella was coupled with multiple HCR (hybridization chain reaction) concatemers and functionalized in a signal structure for lateral flow nucleic acid biosensor (LFNAB) detection. The 16S rRNA was incubated with two specific capture probes and multiple helper probes carrying the same initiator, to unwind its secondary structure and form an "initiators-on-a-string" complex. Through use of the initiators, each target 16S rRNA yielded multiple HCR concatemers tethered to numerous biotins, and numerous streptavidin-labeled gold nanoparticles were introduced on the LFNAB. The limit of detection was 53.65 CFU/mL for Salmonella. Notably, this method has high specificity and applicability for the detection of Salmonella in food and water samples.
Asunto(s)
Técnicas Biosensibles , Nanoestructuras/química , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Salmonella/aislamiento & purificación , Enfermedades Transmitidas por los Alimentos/diagnóstico , Oro/química , Humanos , Nanopartículas del Metal/química , Salmonella/genéticaRESUMEN
In a recent study, we reported a novel assay for the detection of microRNA-21 based on duplex-specific nuclease (DSN)-assisted isothermal cleavage and hybridization chain reaction (HCR) dual signal amplification. The Fam modified double-stranded DNA products were generated after the HCR, another biotin modified probe was digested by DSN and released from the magnetic beads after the addition of the target miRNA. The released sequence was then combined with HCR products to form a double-tagging dsDNA, which can be recognized by the lateral flow strips. In this study, we introduced a 2-OMethyl-RNA modified beacon probe (2-OMe-MB) to make some improvements based on the previous study. Firstly, the substitution of modified probe combined on magnetic beads avoids the fussy washing steps for the separation of un-reacted probes. Furthermore, the modification of 2-OMe on the stem of the probe avoided the unnecessary cleavage by DSN, which greatly reduce the background signal. Compared to the previous work, these improvements save us a lot of steps but possess the comparable sensitivity and selectivity.
Asunto(s)
Sondas de ADN/química , MicroARNs/análisis , MicroARNs/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Humanos , MicroARNs/genéticaRESUMEN
Enterotoxigenic Escherichia coli (ETEC)-induced diarrhea is a complex pathological process, involving ion channel regulation and water efflux. While the mechanism underlying water efflux in ETEC-induced diarrhea is still largely unknown, aquaporins (AQPs) play important roles in transcellular water movement, but their expression profile has not been demonstrated in the murine small intestine. We identified AQP3 expression in the jejunum, but not the duodenum or ileum, using reverse transcription PCR and western blotting. Immunohistochemistry showed that AQP3 localized to the jejunum villi epithelial cells. Using an ETEC-induced murine diarrhea model, we demonstrated that both AQP3 mRNA expression and protein concentration in the jejunum were gradually but significantly decreased over 7 d compared with controls. These results suggested impaired water influx also plays an important role in ETEC-induced diarrhea.
