Detection of EGFR mutations at pM concentration in ten minutes using a microfluidic concentration and separation module

Abstract

Epidermal growth factor receptor (EGFR) mutation detection is now commonly used in the management of cancer patients, particularly those diagnosed with non-small cell lung cancer. Molecular beacon-based sensing is direct and rapid, but its sensitivity is low. Conversely, high-sensitivity detection methodologies based on amplification are robust and sensitive but are limited by relatively require long turnaround times. In this study, we utilized a size-resolved, molecular beacon-based strategy for the rapid detection of EGFR genomic alterations, specifically exon 19 deletions and L858R point mutation. This technology combines a concentration and separation module, which allows us to successfully demonstrate the detection of deletions and point mutations of EGFR in five minutes with a mutant allele sensitivity of 10%. The use of a dual-color detection insures fast detection with a reduced risk of false positives. This work represents a first step toward the fast and specific detection of genetic mutations to improve the management of patients with hard-to-treat tumors.

Fig. 1

Genetic targets definition. The upper panel shows the structure of the EGFR gene. Frequent mutations in the intercellular domain occur in exon 19 and exon 21. We focus on the deletions in exon 19 and the single bp mutation in exon 21, as schematized by red sequences in the bottom panels. All the MBs are flanked by the GCGCGG

Fig. 2

Molecular beacon definition and principle of the detection.(A) The schemes depict the WT and mutated sequence with MBs and the size in bp of the resulting complex in the targeted mode of detection. (B) Same as (A) in the drop-off detection strategy. (C) The plot on the left shows fluorescence intensity as a function of target concentration at a MB concentration of 5 nM. As illustrated in the plot on the right, the fluorescence of unbound MBs is negligible compared to that of MB-target complexes. (D) The micrograph represents the microfluidic concentration module with the shape of a funnel (scale bar = 100 μm). The bottom panel sketches the result of the operation of µLAS with the concentration of the target with one or two MBs at different positions and the flushing of unbound MBs

Fig. 3

EGFR mutation detection.(A) The two fluorescence micrographs represent the signal collected in the funnel (dashed white lines) after three minutes of concentration using 488 nm and 560 nm excitation (left and right images, respectively). (B) The two plots represent the fluorescence intensity profile along the horizontal symmetry axis of the microfluidic chip in the green and the red channel, as outlined on the two images of panel (A). (C) Same as panel (A) in the drop off detection technology. (D) Same as (B) for the two images of panel (C). See the accompanying videos in Supplementary Material