Abstract
Circulating tumor DNA (ctDNA) represents an emerging biomarker of liquid biopsies for the development of precision cancer diagnostics and therapeutics. However, sensitive detection of ctDNA remains challenging, due to their short half-life and low concentrations in blood samples. In this study, we report a new method to address this challenge by integrating cycled enzymatic DNA amplification technique and Au nanoparticle@silicon-assisted surface-enhanced Raman scattering (SERS) technique. We have demonstrated a reproducible identification of a single-base-mutated ctDNA sequence of diffuse intrinsic pontine gliomas (DIPGs), with the limit of detection (LOD) as low as 9.1 fM in the spiked blood samples. This approach can be used to analyze trace amounts of ctDNA in translational medicine for early diagnosis, therapeutic effect monitoring, and prognosis of patients with cancer.
Highlights
Circulating tumor DNA carries genetic information, such as point mutations, methylation, and copy number variations of sequences, of the tumor cells in patients with cancer, simultaneously serving as a diagnostic as well as a prognostic cancer biomarker based on liquid biopsies (Dawson et al, 2013; Weiss et al, 2013; Alix-Panabieres and Pantel, 2016; Wan et al, 2017; Reinert et al, 2018)
The assay is featured with integration of the cycled enzymatic DNA cleavage/amplification and surface-enhanced Raman scattering (SERS) for the sensitive detection of Circulating tumor DNA (ctDNA) (Figure 1A)
The protruding 3’ end in the new double helix can be recognized by Exo III enzyme for the cleavage into nucleotides in a stepwise manner
Summary
Circulating tumor DNA (ctDNA) carries genetic information, such as point mutations, methylation, and copy number variations of sequences, of the tumor cells in patients with cancer, simultaneously serving as a diagnostic as well as a prognostic cancer biomarker based on liquid biopsies (Dawson et al, 2013; Weiss et al, 2013; Alix-Panabieres and Pantel, 2016; Wan et al, 2017; Reinert et al, 2018). We have demonstrated an ultra-high sensitivity (with the LOD of 7.9 fM) and specificity (being able to distinguish a single-base mutation) using this strategy This is a promising approach for the sensitive detection of nucleic acids as a translational tool of ctDNA research. The reaction of cycled enzymatic DNA cleavage/amplification was carried out by mixing 10 μL of P (10 μM), 10 μL of ctDNA in the testing concentrations, 3 μL of Exo III (60U), and 17 μL of the enzyme-reaction buffer at 37◦C for 2 h. H3.3-mutated ctDNA was extracted from the serum according to the extraction procedure of QIAmp DNA Blood Mini Kit. Briefly, 100 μL of serum mixed with H3.3-mutated ctDNA was taken into a 1.5 mL centrifuge tube, 10 μL of protease K and 100 μL of AL buffer were added, and later incubated at 56◦C for 10 min. All intensities at the specified SERS peaks are presented as mean ± SD
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