Liquid biopsy,in particular circulating tumor DNA(ctDNA)analysis,has paved the way for a new noninvasive approach to cancer diagnosis,treatment selection and follow-up.As a crucial step in the analysis,the extraction ...Liquid biopsy,in particular circulating tumor DNA(ctDNA)analysis,has paved the way for a new noninvasive approach to cancer diagnosis,treatment selection and follow-up.As a crucial step in the analysis,the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target.Here,we developed a new generation of microfluidic fluidized beds(FBs)that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15µL/min.We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity,thereby increasing capture efficiency.Taking advantage of this new generation of high-throughput magnetic FBs,we then developed a new method to selectively capture a double-stranded(dsDNA)BRAF mutated DNA sequence in complex matrices such as patient serum.Finally,as proof of concept,ligation chain reaction(LCR)assays were performed to specifically amplify a mutated BRAF sequence,allowing the detection of concentrations as low as 6×104 copies/µL of the mutated DNA sequence in serum.展开更多
基金supported by the European Union Horizon 2020 under Grant 737212:CATCH-U-DNA,Capturing Non-Amplified Tumor Circulating DNA with Ultrasound Hydrodynamicssupported by a PhD grant from ENS Paris-Saclay(L.Alexandre)+1 种基金We would also like to acknowledge the financial support of the CNRS,the Institute Curiereceived the support of“Institut Pierre-Gilles de Gennes”(laboratoire d’excellence,“Investissements d’avenir”program ANR-10-IDEX-0001-02 PSL,ANR-10-EQPX-34 and ANR-10-LABX-31).
文摘Liquid biopsy,in particular circulating tumor DNA(ctDNA)analysis,has paved the way for a new noninvasive approach to cancer diagnosis,treatment selection and follow-up.As a crucial step in the analysis,the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target.Here,we developed a new generation of microfluidic fluidized beds(FBs)that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15µL/min.We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity,thereby increasing capture efficiency.Taking advantage of this new generation of high-throughput magnetic FBs,we then developed a new method to selectively capture a double-stranded(dsDNA)BRAF mutated DNA sequence in complex matrices such as patient serum.Finally,as proof of concept,ligation chain reaction(LCR)assays were performed to specifically amplify a mutated BRAF sequence,allowing the detection of concentrations as low as 6×104 copies/µL of the mutated DNA sequence in serum.
