Surface Enhanced Raman Spectroscopy (SERS) for the Multiplex Detection of Braf, Kras, and Pik3ca Mutations in Plasma of Colorectal Cancer Patients

Abstract

In this paper, we discuss the use of a procedure based on polymerase chain reaction (PCR) and surface enhanced Raman spectroscopy (SERS) (PCR-SERS) to detect DNA mutations. Methods: This method was implemented by first amplifying DNA-containing target mutations, then by annealing probes, and finally by applying SERS detection. The obtained SERS spectra were from a mixture of fluorescence tags labeled to complementary sequences on the mutant DNA. Then, the SERS spectra of multiple tags were decomposed to component tag spectra by multiple linear regression (MLR). Results: The detection limit was 10^-11 M with a coefficient of determination (R²) of 0.88. To demonstrate the applicability of this process on real samples, the PCR-SERS method was applied on blood plasma taken from 49 colorectal cancer patients to detect six mutations located at the BRAF, KRAS, and PIK3CA genes. The mutation rates obtained by the PCR-SERS method were in concordance with previous research. Fisher's exact test showed that only two detected mutations at BRAF (V600E) and PIK3CA (E542K) were significantly positively correlated with right-sided colon cancer. No other clinical feature such as gender, age, cancer stage, or differentiation was correlated with mutation (V600E at BRAF, G12C, G12D, G12V, G13D at KRAS, and E542K at PIK3CA). Visually, a dendrogram drawn through hierarchical clustering analysis (HCA) supported the results of Fisher's exact test. The clusters drawn by all six mutations did not conform to the distributions of cancer stages, differentiation or cancer positions. However, the cluster drawn by the two mutations of V600E and E542K showed that all samples with those mutations belonged to the right-sided colon cancer group. Conclusion: The suggested PCR-SERS method is multiplexed, flexible in probe design, easy to incorporate into existing PCR conditions, and was sensitive enough to detect mutations in blood plasma.

Keywords: PCR; colorectal cancer; gene; mutation; surface enhanced Raman scattering.

Figure 1

Schematic of the PCR-SERS method for the detection of DNA mutations in blood. Genes at Braf15, Kras2, and Pik3ca9 were amplified using mutation non-specific primers. Six mutation types in the three genes were detected using specific probes by probe annealing and subsequent separation. The final SERS measurements were conducted by mixing tags and Ag colloids together.

Figure 2

SERS spectra of the six fluorescence tags of R6G, Cy3, Cy5, TAMRA, ROX, and FAM. The six tags probing mutations V600E, G12C, G12D, G12V, G13D, and E542K, respectively.

Figure 3

SERS spectra results of PCR-SERS applied on the three mixtures with mixing ratios of 1:0:0, 1:1:0, and 1:1:1, respectively.

Figure 4

A) PCR-SERS spectra of the six KRAS wild-type solutions with increasing concentrations (10^-12, 10^-11, 20^-11, 50^-11, 80^-11, and 10^-10 M). B) Linear regression line drawn by the peak intensities of the highest peak at 1314 cm^-1.

Figure 5

SERS spectra of PCR products of plasma samples illustrated using pairs plots. Sub-plots with non-existent mutation combinations are marked as "NA".

Figure 6

A) Heatmap indicating states of six mutation types for all 49 plasma samples. Blue and gray rectangles represent the existence or non-existence of certain mutations, respectively. B) Mutation percentages for the six mutations (V600E, G12C, G12D, G12V, G13D, and E542K) in the 49 plasma samples. C) Venn diagram showing the occurrence and overlap of the mutations at BRAF, PIK3CA, and KRAS

Figure 7

Results of HCA. A) Cluster drawn by all six mutation types (with groupings of TNM stages, differentiation, and cancer position shown on the right). B) Cluster drawn by mutations of V600E and E542K (with grouping of cancer position shown on the right). D: differentiation (poor: brown; moderate: red; well: cyan). S: TNM stages (I: red; II: brown; III: cyan; IV: purple). P: cancer position (left colon: red; right colon: cyan; rectum: brown).