First, purified DNA will be obtained from 2-5 ml of plasma aliquots using commercially available DNA extraction kits. Then, DNA samples will be subjected to an isothermal pre-amplification step to enhance the number of molecules carrying mutations. This pre-amplification step will be carried out into a dedicated thermostatic chamber of Liqbiopsens platform.
Later, amplicon is injected inside the cartridge and processed according to a integrated work-flow. Amplicon DNA is denatured in a pre-treatment stage (1) by melting the dobule-stranded DNA to generate two single strands. In stage (2) single-stranded DNA molecules are transfered to sensor arrays containing DGL probes via a fluidic bypass. Following each amplicon hybridization with their corresponding DGL probe (stage 3), the mutations discriminated is carried out as for stage 4 by using (sensor arrays are immobilized on 4 channels.) novel A, T,C and G biotinylated-SMART Bases (each channel is interogated by individual B-SMART Base). 5) Subsequently, mutations are read out by acoustic reading – biotinylated- SMART Base are detected by specific recognition of streptavidin functionalized nanoparticles. In cases of multi mutations, individual amplified acoustic response will be measured individually on different sensor arrays.