Preparation Of Micro Reactor Identification And Screening Of EGFR Enzyme In Arnebia TKI Applications

Objective:In this research, a micro-reactor based on capillary electrophoresis (CE) was prepared using EGFR as the target for screening, which could be viewed as the combination of biomolecule immobilization and modern micro-analysis. After the preparation of the micro-reactor, it was employed for screening the tyrosine kinase inhibitors (TKI) in Arnebia euchroma extractives. For further research, a separated multi-enzyme micro-reactor was prepared and applied to researching on interactions between diverse components in mixture and their corresponding target enzymes.The technique is expected to be applied to enzyme inhibitor screening, enzyme kinetics, proteomics and related areas.Method:1. Different methods including ionic binding, ionic binding combining with cross-linking agent and covalent binding were used to prepare CE enzyme micro-rector. According to the performance of prepared reactors, the optimized method was chosen to construct the EGFR micro-reactor for screening active ingredients in herb extractives.2. To confirm the effectiveness of the micro-reactor for TKI screening, ATP and Gefitinib were respectively selected as the substrate and known inhibitor, phenylalanine was used as negative control.3. Alcohol quenching method was used to extract alkanin and its derivates, which were separated by CE technique.4. The extractive was introduced into the capillary, and interacted with EGFR, after the specific interaction, the separation was conducted. By comparing peak areas and peak sequence of different components in electrophoretograms, active ingredients could be screened out.5. In further research, different types of enzymes including trypsin, purine nucleoside phosphorylase (PNP) and EGFR were separately immobilized on the inner surface of the same capillary using covalent binding method. The multi-enzyme micro-reactor was combined with CE to conduct online analysis of mixture sample for researching on interactions between components in mixture and different enzymes.Result:1. Two buffering systems were tested, with the same concentration of0.1mol/L and pH value of8.0, the current caused by phosphate buffer was as6times as that caused by borate buffer. Considering the effect of Joule heat, borate buffer was chosen as the experiment buffer.2. The performance of different micro-reactors were tested, the micro-reactor prepared with covalent binding method could be used for more than50times, and the decreasing of combination amount of Gefitinib and EGFR was less than1500. However, the micro-reactors prepared with another three methods could be merely used for less than20times, and the decreasing of combination amount of Gefitinib and EGFR was more than12000.3.0.5mmol/L ATP substrate was introduced into the EGFR micro-reactor, compared with the result obtained from that introduced into the bare capillary, the peak area of ATP decreased by31%, and ADP was produced. Under the same concentration, the peak area of Gefitinib decreased by37%. IC50curve was drawn according to inhibition ratios obtained from different concentrations of inhibitor, and the IC50value of Gefitinib was32.44±0.82μmol/L.4. Alkanin and its derivates were separated by CE with borate buffer (pH8.0) containing20%dehydrated alcohol as running buffer. In the electropherogram five peaks were obtained.5. β-Hydroxyisovalerylshikonin (β-HIVS) extracted from alkanin was found to inhibit the phosphorylation of EGFR.6. According to testing the performance of the micro-reactor, separating immobilizing was more efficient than co-immobilizing, and the effective length of each enzyme reactor was9.64mm. In addition, the separation efficiency could be improved by regulating the pH value of running buffer, and the most appropriate value obtained was9.5.Conclusions:1. Covalent binding method was demonstrated to be a common method for immobilizing different types of protein enzymes, the prepared micro-reactor was effective and stable.2. The immobilized EGFR micro-reactor based on CE was applicable for screening TKI.3. The CE method for separating alkanin and its derivates was established.4.β-HIVS was screened out and found to inhibit the phosphorylation of EGFR.5. Covalent binding method combining with CE technique could be used to prepare separated multi-enzyme micro-reactor for researching on interactions between different components in mixture with their corresponding target enzymes.