Investigation Of Free-flow Eelectrophoresis Chip For Protein And Nucleic Acid Separation

Free-flow electrophoresis is a unique semi-preparative technique used to separate charged analytes.Driven by MEMS technology since 1990 s,free-flow electrophoresis has gradually developed toward the direction of miniaturization.Matrix-free feature of free-flow electrophoresis makes it highly unique over conventional technique,which attributes to its following advantages: continuous operation,mild separation conditions,less sample consumption,an automatic system operated.However,it is not been widely used or even become a method to finish the task of space biological sample processing due to problem of performance stability.Therefore,supported by grants from National special fundation for major scientific instruments,the main purpose herein is to investigate a fundamental strategy for the separation of proteins and nucleic acids via free-flow electrophoresis with stable performance,which will provide a new platform for the purification of biological samples.To achieve the purpose,this study carried via three following aspects:(1)We successfully investigated a glass-based free-flow electrophoresis chip along with performance stability.We firstly designed the geometric structure of chip separation chamber after simulation by COMSOL Multiphysics 3.5a software.Then we researched chip performance stability from two aspects.For one thing,we study it in the terms of gas bubble interference produced by the electrolysis of water.It is helpful to determine the final chip model which can solve gas bubble problem basically.On the other hand,the influence of hydroxymethyl cellulose dynamic coating on the electroosmotic flow was investigated using current-monitoring method in shaped channel glass-based microfluidic chip,which found 0.5%(w/w)-hydroxymethyl cellulose concentration is a suitable condition for inhibition of electroosmotic flow.By applying this condition to the free-flow electrophoresis chip,the results show that hydroxymethyl cellulose dynamic coating turely weakens the hydraulic broadening caused by the electroosmotic flow,and increases separation resolution.(2)Free-flow electrophoresis conditions were optimized.By adjusting electric field strength,sample flow rate and buffer pH,we ultimately determined the appropriate electrophoresis conditions: seperation voltage 81.82V/cm,sample flow rate 3?l/min,buffer pH 5,which is helpful to isolate FITC-BSA: FITC-lysozyme: FITC-pepsin=1:1:1(v: v: v)mixture on the chip.(3)In free-flow electrophoresis chip we successfully isolated and purified proteins and nucleic acids.In chip,we are not only to separate normalized proteins and DNA(BSA,lysozyme and calf thymus DNA),but also achieve the purpose of isolation cell extracts(Jurkat cell total protein and calf thymus DNA mixture).In addition,we used two methods,SDS-polyacrylamide gel electrophoresis and DNA agarose gel electrophoresis,to conduct off-line detection.The results demonstrated that the chip had good performce on the separation of proteins and nucleic acids.In conclusion,this paper has successfully investigated glass-based free-flow electrophoresis chip with good performance stability,which can be used to separate the proteins and nucleic acids.Such research will be expected to provide a favorable support for the treatment of biological samples in clinical medicine,and may even contribute to study space life science.