Absolute Quantification Method For Protein Concentration Establishment And Application Of Electrospray-differential Mobility Analysis-Condensation Particle Counting

Protein quantification is the most basic measurement in biochemical research,and is of great significance in the fields of clinical testing and diagnosis,food safety,and drug analysis.This paper proposes a protein quantification method of high-resolution and high-sensitivity.Electrospray-Differential Mobility Analysis-Condensation Particle Counter(ES-DMA-CPC)is based on the different mobility of charged particles of different sizes in the electric field and obtains different particle sizes and the distributed particles are counted.It has the advantages of no sample pretreatment,high sensitivity,short time consumption,low sample consumption,and wide detection range.In this project,the system is systematically studied through four aspects:the establishment of ES-DMA-CPC,the instrument of ES-DMA-CPC,the parameter optimization of ES-DMA-CPC,and the application of ES-DMA-CPC.The establishment and application of this method not only satisfies the market's demand for quantitative characterization of new technologies,but also has important metrology significance as a potential primary method.The specific contents are as follows:(1)The establishment of ES-DMA-CPC:The evaporation of droplet solvent during electrospray causes the aggregation of particles.This phenomenon is called droplet-induced aggregation effect.We can describe the relationship between the detected aggregates and monomers by mathematical probability statistical analysis and apply it to the measurement of the absolute concentration of protein in the solution.(2)The instrument of ES-DMA-CPC:We set up a set of instruments for absolute quantification of protein concentration.The external sampling system is the FLOW-EZ microfluidic constant flow pump to ensure the stability and accuracy of the liquid injection flow rate.ES is the model 3482 Electrospray Aerosol Generator(TSI,USA),DMA is the model 3082 Differential Mobility Analyzer(TSI,USA),CPC is the model 3788 Water Condensed particle Counter(TSI,USA),and data collection through the Aim program.(3)The parameter optimization of ES-DMA-CPC:Through systematically investigation of the effects of different electrospray voltage,liquid injection flow rate and gas flow rate on particle size distribution,optimizing the conditions for protein quantitative analysis,and using more common silica capillaries(inner diameter 25 ?m,length 4.07 cm).The results show that under the condition of Taylor cone,we can obtain monodisperse particles with small particle size and narrow distribution with the sample injection flow rate as 200 nL/min,the air flow rate as 1.0 L/min,and the carbon dioxide flow rate as 0.1 L/min.(4)The application of ES-DMA-CPC:A fast and accurate assay to determine the absolute concentration of proteins is described based on droplet-induced aggregation effect in electrospray.In this part,we carry out the quantitative application of this method to Bovine Serum Albumin Solution(NIM,BW3627-1),different concentrations of the Bovine Serum Albumin(NIM,GBW09515)and Humanized IgG1?(NIST,RM8671).And through the method comparison,the accuracy,precision and uncertainty of the data analysis,proved that the method is accurate and comparable.