Selective and High Dynamic Range Assay Format for Multiplex Detection of Pathogenic Pseudomonas aeruginosa, Salmonella typhimurium, and Legionella pneumophila RNAs Using Surface Plasmon Resonance Imaging

Abstract

Due to its well-characterized and highly conserved structure, as well as its relative abundance in metabolically active cells, bacterial 16S rRNA sequence plays an important role in microbial identification. In this work, a biosensing strategy has been developed for simultaneous detection of 16S rRNA analytes of three pathogenic bacterial strains: Legionella pneumophila, Pseudomonas aeruginosa, and Salmonella typhimurium. Surface plasmon resonance imaging (SPRi) was used as a detection technique coupled with DNA probe sandwich assemblies and gold nanoparticles (GNPs) for signal amplification. The targets 16S rRNA were selectively captured at the interface of the biosensor by surface-bound DNA probes through a hybridization process. GNP-grafted DNA detection probes were then introduced and were hybridized with a defined 16S rRNA region on the long DNA-RNA sandwich assemblies, resulting in a significant increase of the SPR signal. The results demonstrated the successful implementation of this strategy for detecting 16S rRNA sequences in total RNA mixed samples extracted from the three pathogenic strains at a concentration down to 10 pg mL with a large dynamic range of 0.01-100 ng mL and high selectivity. Since no particular optimization of the probe design was applied, this method should be relatively easy to adapt for quantification of a wide range of bacteria in various liquids.

Implementing Morpholino-Based Nucleic Acid Sensing on a Portable Surface Plasmon Resonance Instrument for Future Application in Environmental Monitoring

“…Interestingly, streptavidin signal amplification alone was successful in reaching a similarly low detection limit (9 pM of a 22-nucleotide long microRNA) in 30 min with the industry standard Biacore X TM instrument [ 14 ]. Another gold-nanoparticle (20 nm) based signal amplification SPR assay reached 0.01 ng/mL detection limit for 16S rRNA of pathogenic bacteria [ 46 ]. A competition assay, where oligo-conjugated gold nanoparticles competed with the target analyte for the binding sites on the SPR surface reached a similar LOD (0.5 nM) for a 22-nucleotide long target, miRNA-200b as our approach [ 31 ].…”

Nanomaterial enabled sensors for environmental contaminants

“…In a recent report, Melaine et al [ 139 ] described the multiplex detection of 16S rRNA from Legionella , Pseudomonas aeruginosa (discussed below) and Salmonella typhimurium . A DNA microarray with capture DNA specific for each target was assembled on a surface plasmon resonance imaging (SPRi) substrate, e.g., gold coated nanoprisms.…”

“…Upon introduction to the RNA ( b (ii)), there is an increase in single ( c (ii)). Finally, gold nanoparticles functionalized with the detection probe (GNP-DP) are introduced and shown to enhance the change in reflectivity (Adapted with permission from Melaine et al [ 139 ]. Copyright 2017 American Chemical Society) …”

“…Similar to Legionella , P. aeruginosa can colonize premise plumbing and has been associated with outbreaks of nosocomial infections in hospitals [ 141 ]. Most of the detection schemes reported for P. aeruginosa focus on whole pathogen detection [ 142 – 146 ] with the work of Melanie et al [ 139 ], discussed above, on 16s rRNA detection being an outlier. In addition, to oligonucleotide recognition elements [ 139 , 142 – 144 ], antibodies [ 145 , 147 ] and bacteriophages [ 146 ] have also been used for specific detection of P. aeruginosa .…”

Label-free detection of nosocomial bacteria using a nanophotonic interferometric biosensor

“…However, most of these SPR assays do not offer a direct detection of the whole microorganism and rather rely on DNA bacterial extraction and identification, 14 or require the use of nanoparticles and enzymes to amplify the sensor signal. 15 Electrochemical, [16][17][18][19] and impedimetric biosensors 20 have been also employed for nosocomial pathogen detection, showing good performance and limit of detection (LOD). However, these approaches also need to amplify the signal with enzymes and/or nanoparticles.…”