Fig. 9: Application of AutoCAR for ctDNA detection from blood plasma (Methods 18, 19, 20, 21). | Nature Communications

Fig. 9: Application of AutoCAR for ctDNA detection from blood plasma (Methods 18, 19, 20, 21).

From: Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing

Fig. 9

A Biosensing performance of AutoCAR-3 in PBS and mouse plasma (n = 3 independent reactions); B AutoCAR-3 calibration curve in mouse plasma (n = 3 independent reactions); C The application of AutoCAR-3 for ctDNA detection in mouse plasma (n = 3 independent reactions); D Plasma samples from patients with advanced cancers harboring the PIK3CA H1047R mutation as determined in tumor biopsies (PIK3CA H1047R + n = 6, and PIK3CA H1047R - n = 4) were subject to detection of circulating PIK3CA mutations in blood plasma using AutoCAR-3 testing. Dashed lines represent the averages of the positive (green) and negative (dark) groups. The feasibility of AutoCAR-3 detection in an alternative body fluid, saliva is shown in Supplementary Fig. 45. E Schematic diagram of AutoCAR-4. The establishment of a Cir-color tag includes the extension of cDNA with 5 nt CCCCC and adding biotin on 3’ end and adding a FAM fluorophore; F The schematic of the colorimetric lateral flow assay (commercial lateral flow strip from Millenia Biotec, with all antibodies pre-integrated) for the detection of Cir-color tag control line: streptavidin; test line: secondary antibody and the application of Cir-color tag-based AutoCAR-4 for ctDNA detection in mouse plasma with a lateral flow assay. The biotin-containing reaction products were captured by streptavidin immobilized on the control line producing color on the control line due to simultaneous presence of Au NPs in these products, as well as FAM and anti-FAM antibodies (anti-FAM Abs). With further flow of the sample, the secondary antibody on the test line captured the anti-FAM antibodies on biotin-free products for colorimetric signal readout. The test line in the CRC LFA (left) shows a darker color compared to the control LFA (right). The background signal in the control LFA is attributed to nonspecific binding or residual Cas12a activation. (Two tails student T test, Error bars represent mean ± SD, *P < 0.05, **P < 0.005, ***P < 0.001, a.u = arbitrary units).

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