Development Of Simple Fabricated Microfluidic Chip And Its Application In Clinical Diagnosis

Objective:The device contains microfluidic channels fabricated from polydimethylsiloxane(PDMS) and double-sided adhesive film (DSA). A major challenge in microfluidicsystem fabrication is to integrate multiple microfluidic components in a simple wayand construct microfluidic systems in a rapid, inexpensive manner. A new technique isdescribed to reduce fabrication time and cost, and shows considerable promise for thetranslation to a rapid point-of-care diagnostic device for the detection of circulatingtumor cells and Escherichia coil O157:H7.Methods:1. To develop simple fabricated microfluidic chip detection system: Designchanges of fluidic channel can be made by ProE software and running a laser cutter,which is applicable to rapid prototyping of microfluidic devices. The microfluidicdevice was assembled with a PDMS layer with holes punched for inlet and outlet port,a DSA and a glass cover slip. After that, we successfully set up a integratedmicrofluidic chip automated reaction system, which used the integrated microfluidicchip as reaction unit, the microvalve controller as the control center, the ten channelinjection pump as the power source, the CCD and conventional inverted fluorescentmicroscope as detection unit.2. Simple fabricated microfluidic chip surface chemistry and immobilized antibodies：In order to achieve the highest antibody immobilization efficiency, we systematicallyinvestigated and evaluated a variety of surface chemical modification and antibodyimmobilized methods. Through the experimental study, we found that reagentconcentration and time will influence the final antibody immobilization efficiency.MPTS-GMBS method is surface modification method, and the streptavidin signalamplification system is antibody immobilization method, which can maintain thehighest activity of antibody.3. Simple fabricated microfluidic chip application in CTCs capture: We utilizedthe simple fabricated microfluidic chip reaction system as the reaction platform, usedMPTS-GMBS-SA method for antibody immobilization, used the microvalve controlthe direction of reagent flow. On this basis, we achieved caputre of CTCs by utilizedimmunofluorescence technique as detection method. In addition, this simple fabricated microfluidic chip detection system also was applied peripheral blood tosimulate blood samples of tumor cells, proved that this system has has practical valveof application.4. Simple fabricated microfluidic chip application in detection of E. coli O157:H7：We utilized the simple fabricated microfluidic chip reaction system as the reactionplatform, used MPTS-GMBS-SA method for antibody immobilization, used themicrovalve control the direction of reagent flow. On this basis, we achieved detectionof E. coli O157:H7without cultivation by utilized immunofluorescence technique asdetection method. In addition, this simple fabricated microfluidic chip detectionsystem also was applied detection of E. coli O157:H7in mineral water, milk sample,proved that this system has has practical valve of application.Results：1. The microfluidic device was successful designed and fabricated. The low cost,easy fabricated microfluidic device has great potential in the application ofpoint-of-care clinical detection.2. MPTS-GMBS method is the best surface modification method, which has thehighest protein fixed efficiency, and the streptavidin signal amplification system is thebest antibody immobilization method, which can maintain the highest activity ofantibody.3. At flow rates of2,4,6and8μl/min, MCF7cell capture efficiencies were (92±3)%,(60±3)%,(24±3)%and (10±3)%, respectively, MDAMB231cell captureefficiencies were (75±3)%,(52±3)%,(22±3)%and (7±3)%, respectively. In addition,this simple fabricated microfluidic chip detection system also was applied peripheralblood to simulate blood samples of tumor cells, At flow rates of2,4,6and8μl/min,cell capture efficiencies were (88±3)%,(50±2)%,(17±3)%and (5±2)%, respectively.4. Under the optimum detection conditions, at flow rates of2μl/min, the totaltesting time and reagent consumption is only30min and60ul respectively, E. coliO157:H7capture efficiencies were (92±3)%. In addition, this simple fabricatedmicrofluidic chip detection system also was applied detection of E. coli O157:H7inmineral water, milk sample, and specificity of86%and93%, respectively.Conclusion：1. The fabricated microfluidics devics is better than convention microfluidic devics, and that has rapid, handheld, low-cost and disposable.2. MPTS-GMBS method and the streptavidin signal amplification system isantibody immobilization method, which can maintain the highest activity of antibody.3. This study successfully built a simple fabricated microfluidic chip reactionsystem and applied in caputre of CTCs. We can rapid capture CTCs from dilutedperipheral whole blood while maintaining high cell viability by fabricatedmicrofluidics devics.4. This study successfully built a simple fabricated microfluidic chip reactionsystem and applied in E. coli O157:H7detection proved that this system has a highvalue of practical application.