Surface-enhanced Raman scattering based ligase detection reaction

Abstract

Genomics provides a comprehensive view of the complete genetic makeup of an organism. Individual sequence variations, as manifested by single nucleotide polymorphisms (SNPs), can provide insight into the basis for a large number of phenotypes and diseases including cancer. The ability rapidly screen for SNPs will have a profound impact on a number of applications, most notably personalized medicine. Here we demonstrate a new approach to SNP detection through the application of surface-enhanced Raman scattering (SERS) to the ligase detection reaction (LDR). The reaction uses two LDR primers, one of which contains a Raman enhancer and the other a reporter dye. In LDR, one of the primers is designed to interrogate the SNP. When the SNP being interrogated matches the discriminating primer sequence, the primers are ligated and the enhancer and dye are brought into close proximity enabling the dye's Raman signature to be detected. By detecting the Raman signature of the dye rather than its fluorescence emission, our technique avoids the problem of spectral overlap which limits number of reactions which can be carried out in parallel by existing systems. We demonstrate the LDR-SERS reaction for the detection of point mutations in the human K-ras oncogene. The reaction is implemented in an electrokinetically active microfluidic device that enables physical concentration of the reaction products for enhanced detection sensitivity and quantization. We report a limit of detection of 20 pM of target DNA with the anticipated specificity engendered by the LDR platform.

Figure 1

Overview of SERS enhanced PCR/LDR detection reaction.

Figure 2

Ethidium bromide-stained agarose gel showing the results of LDR-SERS reaction. Standard marker (lane 1), templates (lane 2), mutant LDR primer (lane 3), LDR product by MT template (lane 4), LDR product by WT template (lane 5), final WT and MT LDR products illuminated without EtBr staining (lanes 6 and 7).

Figure 3

(a) Illustration electroactive microwell device for LDR-SERS-based SNPs detection. Schematic representation of the system showing the lower electrode on the Pyrex glass substrate, the microwell array (diameters of 10 µm and height of 8 µm), and the upper electrically functionalized PDMS gold electrode. Applying the polarity shown in (b) attracts particles and (c) rejects them.

Figure 4

SERS spectra collected on-chip for (a) positive sample containing FMdT-labeled LDR-SERS products by the mutant template (denoted as FMdT-labeled MT), (b) negative sample reacted by WT, (c) control sample containing silver particles and DNA, and (d) background control sample containing silver particles and linker. The concentration of each SNP is 100 pM.

Figure 5

(a) SERS spectra of FMdT-labeled MT in a microwell with the different concentrations of LDR-SERS products. (1) 100, (2) 50, (3) 40, (4) 20, and (5) 10 pM LDR-SERS samples. (b) Plot of peak area at 1610 cm⁻¹ as a function of concentration (R = 0.993). Note that the 10 pM result is omitted from (b) since the concentration was below the limit of detection.