"...our key accomplishment was the demonstration of an eight-plex nonfluorescent DNA detection assay using raman spectroscopy."

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

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

Project Report 2007 - 2008

» Download Project Report 2007 - 2008

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 femto molar (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/gm] 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.

This novel technology, once fully developed, has the potential to detect multiple analytes in a benchtop setting. Further, SERS probes have the potential to be incorporated into living cells to enable real-time monitoring of structural features and electron transfer processes that occur along the DNA helix. This would potentially support probing of DNA damage and splicing mechanisms.

Project Objectives

  • Investigate the effectiveness and efficiency of 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 E. 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 E. coli O157:H7, Campylobacter sp., and Salmonella sp. in milk and water samples.

Project Highlights

We demonstrated that up to eight non-fluorescent raman tags can be chosen with distinct signatures for visual multiplexing utilizing the SERS spectra. The fabrication step has also been optimized and detection sensitivity of up to 1 fM is achievable for the chosen labels. We demonstrated multiplexing of up to eight probes for a chosen DNA sequence. We also showed that hybridization of eight different DNA sequences (depicting eight probes) at one time can be detected. Finally, we developed a strategy to detect DNA sequences in an array (on a glass slide) as well as in a test tube (a one-pot analysis) format.

In summary, our key accomplishment was the demonstration of an eight-plex nonfluorescent DNA detection assay using raman spectroscopy. Steps to standardize the assay for direct detection of target sequences without the PCR simplification step is underway. This technique could be utilized as a slide (lab-on-slide) or tube (lab-on-tube) format for pathogen and disease detection.