Highly adaptable and sensitive FRET-based aptamer assay for the detection of Salmonella paratyphi A.

Here we demonstrate a facile and versatile fluorescence resonance energy transfer (FRET) based aptasensor for rapid detection of Salmonella paratyphi A. The assay shows a detection limit up to 10 cfu·mL-1 with no cross-reactivity with other bacterial species. Less than 8% of inter-assay coefficient variance and recovery rate between 85 and 102% attests the assay reliability. The advantages of FRET-based aptamer assay over the conventional immunoassay formats such as ELISA are the specificity, speed, reliability, and simplicity of the assay. The ssDNA aptamers specific towards pathogenic Salmonella paratyphi A were generated via whole-cell SELEX. The aptamer was conjugated onto quantum dot (QD) that served as the molecular beacon and graphene oxide (GO) was used as a fluorescence quencher. Thus the proposed method enables detection of target pathogen using FRET-based assay. Further interaction of aptamer with pathogen protein DNA gyrase was explored using classical molecular dynamics simulation.

Development of a FRET-based fluorescence aptasensor for the detection of aflatoxin B1 in contaminated food grain samples.

The present study aimed to develop an aptamer-based FRET detection strategy for the specific and sensitive detection of AFB1 in contaminated food grains. The study comprises generation of ssDNA aptamers against AFB1 by whole-cell SELEX and their application in a FRET-based platform utilizing graphene oxide (GO) and quantum dots (QDs). The generated aptamers were characterized to determine their specificity and sensitivity using indirect ELISA where AFB1-OVA was used as a coating antigen. Among the aptamers generated, the ATB1 aptamer showed good reactivity and selectivity against AFB1. This aptamer was further characterized to determine its secondary structure and KD value, which was found to be 5.9 kcal mol-1. The characterized aptamers were conjugated onto Cd/Se quantum dots to develop a fluorimetric system for the detection of aflatoxin B1 using a graphene oxide platform. The presence of graphene oxide quenches the fluorescence ability of the quantum dots due to π-π stacking interactions between the aptamer and GO. Upon target addition, the aptamer forms a complex with aflatoxin B1 thereby restoring the fluorescence intensity. The developed assay shows a linear response from 0.002 µg µl-1 to 0.2 µg µl-1 with a detection limit of 0.004 µg µl-1 for the AFB1 standard toxin and showed no cross-reactivity with other closely related mycotoxins. To validate the reliability of the developed method, several field samples spiked with AFB1 were included in this study and the results obtained were cross verified using a standard commercial AFB1 kit. In conclusion, the developed method may find good utility in routine food testing laboratories for risk assessment of AFB1.