"We are capitalizing on the unique spectroscopic signatures of non-fluorescing molecules as labels to identify specific DNA sequences."

Nanoparticle based DNA multiplexed probes for pathogen detection using confocal raman microscopy

Investigator: Joseph Irudayaraj (Department of Agricultural and Biological Engineering)

Project Report 2008 - 2009

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Project Rationale

The overall goal of this research is to develop a probe fabrication and assay synthesis protocol for multiplex-DNA detection of food pathogens by surface-enhanced raman scattering (SERS) utilizing non-fluorescent, label-containing nanoparticles as DNA probes. Although research on SERS-labeled DNA examination is very active, it is still in its early stages with regard to multiplexing and detecting analytes at low levels. We are capitalizing on the unique spectroscopic signatures (down to ~1 nm resolution) of non-fluorescing molecules as labels (raman tags) to identify specific DNA sequences. Because of the distinct fingerprint of the labels due to SERS, simultaneous detection of multiple DNA hybridizations without separation is feasible at sub femtomolar (fM) sensitivity.

Several aspects are unique to this research. We can use multiplex labeling in one system using a range of non-fluorescing labels. A one-pot platform for detection of food pathogens at sensitivities not afforded by fluorescence methods is possible using our approach. Incorporation of a magnetic separation step will enable the separation of target sequences in complex media. Using non-fluorescent labels (~$10-20/g) for multiplexing is many orders cheaper than fluorescent labels (~$10-20/mg). Furthermore, the choice of SERS labels is enormous (over 1,000 labels) and extremely sensitive, and single-molecule identification has been reported. This implies that eventually the detection can be accomplished without the amplification step.

Project Objectives

  • Investigate the effectiveness and efficiency of two widely available fluorescent and five cheaper non-fluorescent dyes as raman labels to be used as SERS tags.
  • Synthesize SERS-DNA probes for detecting species-specific DNA sequences of Escherichia coli O157:H7, Campylobacter sp., Staphylococcus aureus, Listeria monocytogenes, and Salmonella sp. as targets.
  • Develop a one-pot multiplex detection system using the optimized SERS-DNA probe to simultaneously detect genes related to pathogens.

Project Highlights

Using the SERS method, we developed an eight-plex strategy to detect up to eight DNA interactions simultaneously. The raman labels we chose were non-fluorescent in nature; hence the detection cost is about 1,000 times cheaper than conventional fluorescence methods. In addition to detection, quantification of gene fragments was also possible at a level of sensitivity reaching 1 fM. This was accomplished in an array format. We also developed a tubeformat eight-plex detection scheme using non-fluorescent labels. We used this scheme to detect oligonucleotides depicting the three key pathogens. This proof-of-concept study demonstrates that the methods we developed can be used to detect and quantify genetic fragments extracted from multiple pathogens.