"Our key accomplishment this year is the demonstration of an eight-plex, non-fluorescent 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 2006 - 2007

» Download Project Report 2006 - 2007

Project Rationale

The overall goal is to develop a framework with probe fabrication and assay synthesis protocols 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. Our team is 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 hybridization without separation is feasible at sub femto molar (fM) sensitivity.

There are a number of unique aspects to this project. We can use multiplex labeling in one system using a range of non-fluorescing labels. A single 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. Use of non-fluorecent 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 1000 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

  • Optimize the SERS effect of dye/gold particle size and excitation wavelength and concentrations with multiplexing. We have 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 target.
  • Fabricate SERS-tagged DNA probes for each of the five target pathogens and multiplex demonstrations. Multiplexing of up to eight probes has been demonstrated for a chosen DNA sequence. We have also demonstrated that hybridization of eight different DNA sequences (depicting eight probes) at one time can be detected.
  • Design and implement an analysis assay for pathogen detection using SERS DNA probes.

Project Highlights

Our key accomplishment this year is the demonstration of an eight-plex, non-fluorescent DNA detection assay using Raman spectroscopy. We are now undertaking steps to standardize the assay for direct detection of target sequences without the PCR amplification step. The developed technique could be utilized as a slide (lab-on-slide) or tube (lab-on-tube) format for pathogen and disease detection.