Research On Electrokinetic Stacking Method Of Paper-Based Analysis Device For Purification And Detection Of Protein

Paper-based analysis device(PAD),as a new low-cost microfluidic analysis and detection platform,is very suitable for rapid detection of samples.Therefore,it has attracted widespread attention in the fields of medicine,ecological environment,and food safety.Online pre-concentration can effectively enhance the sensitivity of the PAD.Field amplification stacking and isoelectric focusing are common electrokinetic stacking methods.It is of great significance to introduce them into PAD to improve its detection ability.In the first chapter,the principle,development and application of common electrokinetic stacking methods in PAD are introduced,field amplification stacking and isoelectric focusing are highlighted.The high-salt sample processing method is briefly introduced,and the application of 2-DE,affinity chromatography and Off-Gel in proteomic analysis pretreatment is introduced.The second chapter introduces a new field amplification stacking method,which generates an electric field gradient through the structural design of the paper-based fluidic channel and reduces the limitation of the conductivity of the sample media.By directly loading the sample onto the PAD,the transfer rate is effectively increased,and 93.5%of the target component in a small sample is stacked in the stacking region.The limit of detection(LOD)of 6.5 mg·L-1 was obtained,and the linear quantitative range was 10～100 mg·L-1(R2=0.991).The LOD of colorimetric detection of human serum albumin(HSA)was 2 orders of magnitude improved.The colorimetric detection of microalbuminuria(MAU)by mobile phone imaging successfully demonstrated the ability of this online pre-concentration method to analyze target components in physiological samples.In the third chapter,the double gradients(E/pH double gradients)could be established on a paper fluidic channel through the design of the electrolyte.The protein components in the high salt media can not only be stacked on the double gradients,but also be separated from other types of components to achieve desalination and purification.The method is applicable to the colorimetric detection of HSA with a linear quantitative range of 10～300 mg·L-1(R2=0.99)and a LOD of 4.9 mg·L-1.This method was used to detect the MAU in the urine of diabetic patients,and there was no significant difference between the test results and the clinical results(immunoturbidimetry).This work demonstrates the potential of paper fluidic channel for purification of target components in high-salt samples and effectively improves the sensitivity of paper fluidic channel in Point of Care Test(POCT).The fourth chapter is based on the previous carrier ampholytes free isoelectric focusing(CAF IEF)method,with the rapid pretreatment of Hela lysates,the potential of CAF IEF in complex proteome analysis was demonstrated,which provides a technical approach that different from the traditional sample purification and pretreatment methods.The method not only require no CA to perform quasi-liquid rapid pre-separation of proteins based on the isoelectric point,but also has a certain sample purification function,thereby becoming a low-cost pretreatment method for complex protein samples,which is expected to be applied in the discovery of biomarkers and accurate diagnosis in daily clinical practice.