Research On The Enrichment Of Circulating Tumor Cells Based On The Integrated System Combining Inertial Separation With Immune Recognition

As a significant biomarker of liquid biopsy,circulating tumor cell(CTC)has broad prospects in clinical diagnosis,early screening,and targeted drugs development.However,the extremely low abundance of CTCs in peripheral blood emphasizes the necessity of efficient CTC enrichment before further clinical diagnosis and treatment.Present CTC enrichment systems usually rely on a single separation strategy,which cannot meet the requirements of recovery and purity at the same time.Based on this demand,an integrated CTC enrichment system based on inertial separation and immune recognition was proposed in this paper.Through the integration of the inertial microfluidics and immune recognition,the system could realize high throughput,high recovery,and high purity simultaneously.To solve the key problem in the integrated system,this paper was demonstrated as follows:To increase the stability of the separation system,a spiral inertial microfluidic chip with periodic expansions was proposed in this paper,and a wider range of throughput for cell separation from 400μL/min to 1000μL/min could be realized when the expansions were introduced into the spiral inertial microchannel,which could overcome the strong dependence of the chip structure and flow rate in the traditional spiral inertial microfluidics.It is proven that the inertial separation chip can eliminate the majority of blood cells for subsequent immune recognition on the premise of high recovery rate of CTCs.To overcome the low capture efficiency caused by the poor antibody immobilization in immune separation,a layer-by-layer self-assembled monolayer strategy was proposed.The Au-S bond between Au NPs and mercaptan was used to immobilize the specific antibody on the chip surface spontaneously and orderly,realizing the specific capture with the recovery of 84.51%.Meanwhile,the twin-layer vein-shaped microstructure in the chip could ensure the full contact between the cells and the chip surface,enhancing the capture efficiency of CTCs.In view of the problem that it is difficult to obtain totally complete cells after CTC enrichment for subsequent clinical research,the trypsin digestion method was utilized to realize the non-invasive release of captured cells.Compared with mainstream cell release strategies through cutting off the coupled molecular chain of antibodies,trypsin digestion could completely remove the coupled antibodies and other molecules by digesting the antigen on the cell surface and realize the release efficiency of 92%.It is proven that the digested antigen could be completely recovered through gene re-expression,which is proven to be harmless to cells.Aiming at the limitation of current CTC enrichment method in hardly guaranteeing high throughput,high recovery,and high purity simultaneously,an integrated cell separation system was proposed combining inertial separation and immune recognition through on-chip reduction of velocity and binary tree structure design.Meanwhile,a miniaturized cell sorting instrument was designed and developed to reduce the loss and contamination of rare cell sample induced by secondary injection,realizing the efficient enrichment of CTCs with high throughput,high recovery,and high purity simultaneously.