Research Program
Biomolecular Devices and Analysis

Objectives
In this research we propose a new approach to the molecular diagnosis of cancer through the development of a Surface Enhanced Raman Scattering (SERS) enhanced Ligase Detection Reaction (LDR) for Single Nucleotide Polymorphism (SNP) detection.  Of the many technologies available for SNP diagnostics, Real-Time PCR (RT-PCR) is one of the most appropriate for clinical analysis, requiring the smallest amount of sample preparation and providing the most rapid results.  RT-PCR and similar solution phase reactions however are limited in the degree to which they can be multiplexed by spectral overlap between the florescent dyes that are used.  The LDR-SERS based SNP detection reaction and microfluidic chip (Figure 1) to be developed here avoids this problem by detecting the Raman fingerprint of the dye rather than the florescence emission.  Briefly the reaction uses two LDR primers, one of which contains a Raman enhancer and the other a dye.  In the presence of the SNP the primers are ligated and the enhancer and dye are brought into close proximity enabling the dye’s Raman signature to be detected.  Since every dye has a unique Raman signature, spectral overlap is avoided and the number of SNPs which can be screened for in parallel is dramatically increased.

Methods
In our proposed scheme, different pairs of LDR probes will be designed where one member of the pair has a common SERS enhancer and the other has a fluorophore.  The overall strategy is outlined in Figure 2.  For simplicity a single set of PCR products are presented with two LDR probes.  Each LDR probe uses a common Raman enhancer and each of the accompanying LDR probes located upstream is labeled with a fluorophore.  Since SERS spectra are unique for each fluorophore or Raman-active dye, we will use a unique label for each target sequence. Due to the high information content of SERS spectra, we will be able to discriminate between as many as 12 different Raman-active dyes.

Summary
In this program we propose the following two research thrusts as described below.

Research Thrust #1 – Developments towards a 12-Plex SERS Based LDR Reaction for SNP detection.  In our first research thrust, target-specific PCR primers and LDR probes will be designed, synthesized and tested in a single assay format. The design and synthesis of LDR probes will be explored by varying their composition and placement of the dye and Raman enhancer.  As described below we have already demonstrated a single-plex reaction (Figure 3) and our goal here will be to extend this to demonstrate the screening of as many as SNPs in parallel as possible.  Our goal will be to eventually get to 12, which will represent at least a doubling of the state of the art.

Research Thrust #2 – Develop optically resonant electroactive microwells for on-chip SERS detection.  Acquisition of spectral information from the LDR-SERS reaction is more challenging that the intensity measurements made for florescence detection.  In this research thrust we will develop a unique microfluidic system to localize the sample and electromagnetic energy so as to reduce detection time to the same level as that possible with RT-PCR.  In the second research thrust we propose to develop a new approach to chip based SERS detection based on combining optical ring resonators with our electroactive microwells

Goals & Accomplishments

• Developments towards a 12-Plex SERS Based LDR Reaction for SNP detection
• Develop optically resonant electroactive microwells for on-chip SERS detection.