Electrokinetic Enrichment,Separation And Clean-up Of Protein Sample On Microfluidic Paper-Based Analytical Device

Paper-based microfluidic analytical devices(μPADs)have drawn wide attention in the point-of-care test(POCT)of diseases due to their advantages such as low cost,portability,easy to use,and easy to fabricate.Proteins are rich in information for disease diagnosis,but many of them are from samples with relative low concentration level and complex matrix.To improve the detection sensitivity and selectivity of the paper-based analytical device for the target proteins,electrokinetic pretreatment methods of proteins were investigated in this work on μPADs.In the introduction section,the main features and developing trends of μPADs were introduced,and the fabrication methods,detection methods and electrokinetic sample pretreatment methods for μPADs were summarized.In the experimental section,firstly,a paper-based isoelectric focusing device was implemented for simultaneous separation and concentration of proteins.After selection of the appropriate paper substrate material and pre-treatment with hydroxyethyl cellulose,isoelectric focusing of proteins on the paper fluidic channel was successfully demonstrated with the aid of carrier ampholytes.Then,this method was used for the separation and detection of human serum albumin(HSA)and glycated hemoglobin(HbA1c).The colorimetric analysis showed that the concentration of HSA was 40 mg/mL and the hemoglobin sample contained 5.6%HbAlc.In addition,the separation fractions of this IEF were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and matrix-assisted laser desorption ionization-time of flight-mass spectrometry(MALDI-TOF-MS).Secondly,based on the principle of pH-Mediated field amplification stacking,an electric field gradient was established on the paper channel by the neutralization titration reaction between OH-and Tris-HCl.Due to field amplification stacking effect,anions in the high salt sample matrix were well concentrated and the enrichment factor was about 100.This electrokinetic enrichment method was applied to concentrate and analyse urine albumin and Cr(VI)in electroplating wastewater.The measured concentration of albumin in the adult urine with nephropathy was 0.84 mg/mL.Finally,a sharp pH gradient was established based on the moving neutralization reaction boundary formed by the neutralization titration reaction between OH-and Tris-HCl on the paper fluidic channel,with which simultaneous enrichment and separation from anions,cations and neutral molecule of proteins were demonstrated.With this method,proteins can be concentrated about 10 folds.The proposed method of electrokinetic enrichment,separation and clean-up of proteins in this paper can not only be used to improve the detection sensitivity of paper-based analytical device,but also can be used as a sample pretreatment method coupled with other analytical devices.