Study On CCK-8 Cell Proliferation Kit And Bioluminescence Detection Technology Of Ethylenediamine

There are two parts divided in this thesis:In the first part,we explored the formulation and preparation of the CCK-8 kit,which was developed by the Dojindo Molecular Technologies,Inc.,and to apply it to the cytotoxicity test.Based on these results,we designed and synthesized three new WST-8 derivatives and evaluated their optical properties.In the second part,we designed and synthesized the first bioluminescent probe for the detection of ethylenediamine.This probe has high selectivity and sensitivity,low toxicity and is capable of detecting ethylenediamine in aqueous solution,cells and small animals.In summary,these results may provide a new way to the advancement and development of the biomedical diagnostic reagents.Part Ⅰ(Study on Methods for Determination of Cytotoxicity):The drug cytotoxicity test,that is evaluated by comparing the changes in the number of cell populations of the experimental group and the control group to calculate the survival rate of the cells.The CCK-8 kit(Cell Counting Kit-8)is a colorimetric determination assays of cell proliferation or drug toxicity,which has highly sensitive,non-radioactive.So far,CCK-8 reagents are expensive and mainly rely on import.Herein,we studied the synthetic route of WST-8,the main active composition of CCK-8 reagent.NBS(N-Bromosuccinimide)was used as the oxidant in the final step of the synthetic route,and the total yield was increased to 40%.Furthermore,the formulation and preparation method of CCK-8 developed by the Dojindo Molecular Technologies,Inc.was well studied.Finally,three new structural derivatives of WST-8,named coumarin tetrazolium salts,were synthesized and the UV-visible absorption spectra of these salts and corresponding formazan compounds preliminarily obtained.Formazan,the reduced form of coumarin tetrazolium salts had strong absorption capability on visible area,and the maximum absorption wavelength was between 420 and 450 nm.The result suggested that these new compounds have the potential to become new biosystem redox indicators.The second part(Development of ethylenediamine bioluminescent probe):Bioluminescence is a specific protease(luciferase)in the body to catalyze the oxidation of specific chemicals(luciferin).Biochemical energy is almost completely converted into chemiluminescence of light energy.Compared with fluorescence imaging,bioluminescence imaging has the advantages of high biocompatibility,no need for external excitation light,strong targeting,and high sensitivity.Based on the firefly luciferase bioluminescence system,we developed the first bioluminescent probe for the detection of the toxic substance ethylenediamine.We first protected the amino group of aminoluciferin with 3-aminophthalic anhydride,preventing the probe from being recognized by firefly luciferase.If the aqueous solution or the biological sample contains ethylenediamine,the probe can be aminated to release the substrate firefly luciferin.Eventually,firefly luciferin binds to luciferase to produce bioluminescence.The probe has high selectivity and sensitivity,low toxicity and ability to detect ethylenediamine in aqueous solution,cells and small animals.It is a promising ethylenediamine detection probe,which is a highly sensitive and non-radioactive colorimetric assay for determining the number of viable cells in cell proliferation or cytotoxicity.