Research On The Deterministic Lateral Displacement Structure Microfluidic Chip Used In Rapid Isolation Of Circulating Tumor Cells

Objective:To design and fabrication a microfluidic device with deterministic lateraldisplacement （DLD） arrays allowing rapid circulating tumor cells separation andenrichment from diluted peripheral whole blood while maintaining high cell viability,establish a new size-based circulating tumor cells separation and enrichment method.Methods:1. Microfluidic device design: According to our previous research and the theorydeveloped by Inglis et al.,we designed two types of microfluidic device with differentDLD arrays,circular and triangular post arrays.The critical particle size, the radius andgaps of microposts can be calculated.2. Device fabrication：Microfluidic devices were fabricated using standardphotolithography and soft lithography. Negative photoresist SU8-3025was used tofabricate the master on silicon wafer with a photomask. Next, the patterned siliconwafers were silanized with chlorotrimethylsilane to facilitate PDMS mold release.PDMS prepolymer mixed with curing agent （5:1w/w ratio） was poured onto thesilicon wafer and curried at80℃for1hour. Finally holes were punched for inletand outlets and the PDMS mold was bonded to glass slides after oxygen plasmatreatment.3. Microfluidic CTCs isolation：To determine the isolation efficiency of ourdevice, cancer cells （MDAMB231cells, MCF-7cells, A549cells, HEPG2cells,KYSE150cells） were spiked into phosphate buffer saline （PBS） at105cells/mL forcircular post arrays DLD chip. MDAMB231cells and MCF-7cells for triangular postarrays DLD chip. The inlet with cell suspension solution was connected to a syringepump which controlled the fluid velocity from0.03mL/min to2mL/min.The cellmovement in the microchannels was observed under microscope and recorded by ahigh speed CCD camera. The concentrated cells were collected in the middle narrowoutlet, which was connected to another syringe pump controlling the output flow. Bymeasuring the cell concentration and volume of the output, we can calculate the isolation efficiency with the following equation:Isolation efficiency=C₁V₁/（C₁V₁+C₂V₂）where C1and V1are the CTCs concentration and solution volume in thecollection outlet, C2and V2are theCTCs concentration and solution volume in thetwo waste collection outlets.4.Blood specimen processing: All blood samples were collected intoEDTA-containing tubes and were processed within12hours. As blood has highviscosity, it was diluted10times with physiological saline. Cancer cells were firststained by Vybrant DyeCycle Green to be discriminated from other blood cells byfluorescence microscope. Next cancer cells were spiked into the diluted bloodspecimens at¹04cells/mL. After CTCs isolation, the cell isolation efficiency wasfinally calculated.5.Using ANSYS software and Gambit software analysis to further explore therelationship between flow rate, DLD array shape, and the isolation efficiency.Results：1. Circular and triangular post arrays DLD microfluidic devices were successfuldesigned and fabricated. Cancer cells can be collected in the narrow channel notappear in two broad outlet channels.2. At low flow rate, all cancer cells and two types DLD microfluidics devics canachieved high isolation efficiency. In circular post arrays DLD chip, when flow rate is0.03mL/min, MCF-7and KYSE150have a high isolation effciency（99%and98%）,MDAMB231, A549and HEPG2also90%. When flow rate is100μL/min, theisolation effciency of MCF-7and KYSE150is50%, MDAMB231, A549and HEPG2about40%. When the flow rate is1000μL/min all of cells have low isolationeffciency （just20%）. The cell isolation efficiency decreased significantly with theincrease of flow rate. The isolation efficiency of circular DLD array decreasedsignificantly at high flow rate while triangular DLD array was able to keep the highisolation efficiency. In triangular post arrays, when flow rate is30μL/min, MCF-7cells’s isolation effciency is100%and MDAMB231is99%, when flow rate is100μL/min, the isolation effciency of MCF-7cells is97%, MDAMB231is95%.When flow rate is2000μL/min, the isolation effciency of MCF-7still95%, but MDAMB231is64%. The larger cells have higher isolation efficiency then smallcells.3. Triangular DLD array can get a high isolation efficiency, about95%. By thismicrofluidics devics we can rapid isolationg CTCs from diluted peripheral wholeblood while maintaining high cell viability.4. The cell deformation can influence the cell isolation efficiency of DLDdevice.Cell deformation resulted in the decrease of cell effective radius. If theeffective radius is less than the critical particle size of the DLD array, cells cannot beseparated by the device. Cell deformation explains why the cell isolation efficiency ofcircular DLD array decreased with the increase of flow rates in our study.Fortriangular post array, the contact area between a cell and a post is a point rather than aplane, it is difficult for cells to deform.The relationship between flow rate, DLD arrayshape, and CTCs deformation is further explored.Conclusion：1. The separation enrichment effect of triangular DLD array is better thancircular DLD array.2. we can rapid isolationg CTCs from diluted peripheral whole blood whilemaintaining high cell viability by triangular DLD array microfluidics devics.3. The relationship between flow rate, DLD array shape, and the isolationefficiency is further explored by shear stress and the deformation of cells.4.This research still has many places need to improve. The tumor cellconcentration in peripheral blood is higher than CTCs in peripheral blood. After theseparation,There are still a lot of blood cells. Did not combined with other techniquessuch as antibody based immuno-binding methods for post-capture processing.