Design of peptide nucleic acid probes on plasmonic gold nanorods for detection of circulating tumor DNA point mutations

Abstract

Here we present a gold nanorod-based platform for the sequence-specific detection of circulating tumor DNA (ctDNA) point mutations without the need for amplification or fluorescence labeling. Peptide nucleic acid probes complimentary to the G12V mutation in the KRAS gene were conjugated to gold nanorods, and the localized surface plasmon resonance absorbance through the sample was measured after exposure to synthetic ctDNA at various concentrations. Each step of the reaction was thoroughly controlled, starting from reagent concentrations and including conjugation, sonication, and incubation time. The platform was evaluated in both buffer and spiked healthy patient serum, demonstrating a linear working range below 125 nanograms of ctDNA per milliliter solution, and an effective limit of detection of 2 nanograms of ctDNA per milliliter. A clear distinction between mutant and wild type synthetic ctDNA was also found using this platform. In order to improve upon the selectivity of the sensor, a DNA hybridization simulation was performed to understand how the addition of mutations to the peptide nucleic acid probe could enhance the selectivity for capture of mutant over wild type sequences. The top candidate from the simulations, which had an additional mutation two base pairs away from the mutation of interest, had a significant impact on the selectivity between mutant and wild type capture. This paper provides a framework for sequence-specific capture of ctDNA, and a method of improving selectivity for desired point mutations through careful probe design.

Keywords: Circulating tumor DNA; Liquid biopsy; Localized surface plasmon resonance (LSPR); Nanorods; Nanosensor; Point mutation; Surface conjugation.