Abstract
Detection of single-nucleotide polymorphism (SNP) in circulating tumor DNA (ctDNA) is challenging because of the large DNA fragmentation (∼150 nt) and the strong background of normal cell free DNA (cfDNA). Here we developed a rapid centrifugation-assisted colorimetric assay using gold nanoparticles (AuNPs) coupled with isothermal amplification to detect a SNP (G to C mutation) in KRAS, p.G13D in ctDNA. Compared to conventional AuNP aggregation assays, our assay contains four unique design concepts. Firstly, a centrifugation step is introduced at the end of the reaction that significantly enhances the colorimetric readout by providing visually distinct precipitation for the SNP ctDNA. Secondly, to achieve a fast turnover rate for clinical pM demand, a "critical linker concentration" concept is introduced to the assay. Thirdly, in order to achieve an unambiguous differentiation of the SNP ctDNA from wild type cfDNA and the control sample without DNA, a "color code conversion" strategy is employed, where a complementary sequence of the linker DNA is introduced to manipulate the AuNP aggregation. Finally, ethylenediaminetetraacetic acid is used for enzyme inactivation only at room temperature while stabilizing the AuNP solution from unwanted aggregation. Our assay coupling two amplification strategies (isothermal amplification and centrifugation-assisted assembly) is capable of both quantitative and qualitative differentiation of SNP in ctDNA of ∼150 nt at a clinically relevant concentration and 67 pM limit of detection and in the presence of 99% normal cfDNA background. This assay can be used for point-of-care colon cancer diagnosis and prognosis with a fast turnover time (<2 h).