This Small Business Innovation Research (SBIR) Phase I project proposes to develop an entirely new platform for the molecular diagnostics of cancer exploiting a new Surface Enhanced Raman Scattering (SERS) enhanced Ligase Detection Reaction (LDR) and an optically active microfluidic chip. Since every dye has a unique Raman fingerprint, the number of single nucleotide polymorphisms (SNPs) that can be screened for in parallel is dramatically increased due to lower spectral overlap. The goal for the Phase I work is to develop a set of target-specific LDR probes in a 6-plex format that would match the current state-of-the-art. For future work, we will develop an assay for a 12-plex format that will double the number of targets that can be screened in one sample using this fluorescence-based technology. In parallel with the development of the screening reaction, we will create a novel "optofluidic" chip that addresses the challenges with on-chip Raman signal detection. The primary advantage of the proposed approach is that it avoids the fundamental limitation of the existing state-of-the-art, Real-Time Polymerase Chain Reaction, by detecting the Raman fingerprint of the dye rather than the florescence emission.
The broader impact/commercial potential of this project is the ability to screen for a greater number of single nucleotide polymorphisms (SNPs) without sacrificing speed and ease-of-use, which may enable more rapid molecular diagnosis of cancer and other genetic diseases. In some cases, SNPs are biomarkers implied in the cause of cancer while in others they represent markers indicative of an increased risk of cancer. In either case, SNPs have been shown to be good biomarkers for many classes of cancer and have further been shown to correlate with various clinicopathological features of different cancer subtypes. Of the many technologies available for SNP diagnostics, Real-Time PCR (RT-PCR) is the most appropriate for clinical diagnosis in that it requires the smallest amount of physical sample and sample preparation; however, it is limited in terms of the number of SNPs that can be screened at one time. The approach we propose will potentially double the number of SNPs that can be screened in a single sample. This advantage is particularly important in cases where the biopsied sample is not homogeneous.
Intellectual merits of proposed activity During the proposal we developed an entirely new platform for the molecular diagnostics of cancer exploiting a new Surface Enhanced Raman Scattering (SERS) enhanced Ligase Detection Reaction (LDR) and a Raman enhanced microfluidic chip. The primary advantage of the approach we proposed is that it avoids the fundamental limitation of the existing state of the art, Real-Time Polymerase Chain Reaction, by detecting the Raman fingerprint of the dye rather than the florescence emission. Since every dye has a unique Raman fingerprint, the number of single nucleotide polymorphisms (SNPs), which can be screened for in parallel is dramatically increased due to lower spectral overlap. Simultaneous with the development of the screening reaction, we will create a novel SERStrate active chip which addressed the challenges with on-chip Raman signal detection. The proposed device exploited simultaneous physical concentration of the sample using electro active microwells and SERS enhancement using the patented SERStrate enhancement. Results The Phase I research showed six of the seven SERS probes produced reliably readable spectra both individually and in mixing experiments with 2-3 probes. Three to nine replicates were performed for each, with corresponding negative controls. The positive spectral signals were clearly distinguishable from negative reaction spectral signals. At no point did a negative control demonstrate a positive spectrum, confirming the high level of specificity of the LDR reaction as detected by SERS. Given that even the small sample sizes we collected allow us to distinguish most of our compounds, we are confident that expanding data collection will distinguish all compounds with a high degree of accuracy and specificity without any manual manipulation of the spectra. Furthermore, these results indicate that it should be possible to reliably identify at least 12 different fluorophore spectra. Broader impacts/commercial potential Cancer is the uncontrolled growth of cells an event which is caused by a series of cellular events. In some cases single nucleotide polymorphisms (SNPs) are the implied cause of cancer while in others they represent well defined molecular markers indicative of an increased risk of cancer. In either case, SNPs have been shown to be good biomarkers for many classes of cancer and have further been shown to correlate with various clinicopathological features of different cancer subtypes. Of the many technologies available for SNP diagnostics, Real-Time PCR (RT-PCR) is the most appropriate for clinical diagnosis, requiring the smallest amount of physical sample and sample preparation, but it is limited in the number of SNPs which can be screened at one time. The approach we propose here will at least allow us to double the number of SNPs which can be screened for in a single sample. This advantage is particularly important in cases where the biopsied sample is not homogeneous. The ability to screen for a greater number of SNPs without sacrificing on rapidity and ease could enable more rapid molecular diagnosis of cancer and other genetic diseases.
