Application Of Electron Spin Resonance In Immunoassay And Antioxidant Capacity Measurement

In this dissertation,we combined electron spin resonance(ESR)technique and analytical chemistry research.There are mainly two aspects.Firstly,electron spin resonance spectroscopy was applied to immunoassay.With rabbit immunoglobulin G(Ig G)as the analyte,we developed two immunoassay methods based on electron spin resonance.In the 1st method,iron oxide nanoparticle was labeled to antibody,and Fe3+ in the nanoparticle was probed for ESR signal.The 2nd method was based on enzyme-linked immunosorbent assay(ELISA).In the 2nd method,2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt(ABTS)was used as substrate and organic free radicals generated from ABTS was detected by ESR.In both methods,the quantitative relationship between the ESR signal and the concentration of analyte was established.Secondly,we developed an ESR method for determining the antioxidant capacity of small molecules based on Cu2+ reduction.We applied this method to the determination of the antioxidant capacity of thiol-containing compounds and their pharmaceutical formulations.Also the method was applied to the measurement of some fruits' antioxidant capacity.With iron oxide nanoparticles of two different diameters as probe and with rabbit Ig G as the analyte,we developed a new competitive immunoassay method based on ESR technique.When 10 nm nanoparticle was used as probe the sensitivity was 1.81 ?g m L-1,whereas when 30 nm nanoparticle was used the sensitivity was 0.014 ?g m L-1.Compared with other existing immunoassay methods,the antibody linking time used in the present method is the shortest(1.5 h).The immunoreaction time used in the present method is close to that in surface plasmon resonance(SPR)method,which is shorter than those used in other methods.When the present method was applied,the immunoreaction was performed in the very cheap plastic tube and thus the simultaneous treatment of a large amount of samples was easier.Compared with most optical spectroscopic methods,such as ELISA and fluorescence methods,there are no background signal for IONPs in most biological materials and no color interference for the ESR measurements.In addition,we found that the measurement samples could be stored for at least five days and no change in either signal shape or intensity could ever be observed.This method could be valuable for the reexamination of clinical samples.Based on the capability of free sulfhydryl group in reducing Cu2+ ion,the ESR spectroscopy was applied to the quantification of the antioxidant capability of thiol-containing compounds.With this method,the antioxidant capacity of eight thiol-containing compounds,including reduced glutathione,N-acetyl-L-cycsteine,methimazole,captopril,tiopronin,1,4-dithioerythritol,2,3-dimercapto-1-propanol,and cystine,was determined.The method was also applied to the quantification of N-acetyl-L-cycsteine,methimazole,captopril,and tiopronin in their pharmaceutical formulations.Trolox equivalent antioxidant capacity(TEAC)was used to quantify the antioxidant capacity of the thiol-containing compounds.The antioxidant capability of the eight compounds obtained by ESR was proved to be highly correlated to the number of free sulfhydryl bonds existing in the molecules.Cystine does not contain free sulfhydryl group and the TEAC value was close to zero.The TEAC values for the five compounds with only one sulfhydryl group were close to each other.Their average value is 0.53,which is approximately one half of the average TEAC value(1.03)for 1,4-dithioerythritol and 2,3-dimercapto-1-propanol with two groups.In the case of methimazole,the number of the sulfhydryl group derived from traditional Ellman assay is 0.01,which is too low.The TEAC value for methimazole obtained by cupric reducing antioxidant capacity(CUPRAC)method is 1.88,which is too high compared with the normal TEAC value(0.5)for one sulfhydryl group.The present method supplied reasonable TEAC values for all eight thiol-containing compounds.The recoveries obtained by the present method were found to be 95.3-103.7%.It was indicated from the analytical results that the present method is suitable for the analysis of practical drug samples.With rabbit Ig G as the analyte and with ABTS as the substrate of horse radish peroxidase(HRP),we developed an ELISA based on biotin-avidin-system and the experimental conditions were studied.Also the feasibility of ESR to be used as the detector for ELISA was discussed.When the ratio of antibody to biotin was increased from 1:10 to 1:30 and 1:100,the slope of the standard curve increased and the signal was enhanced.When the ratio of HRP-biotin to avidin was increased from 1:1 to 1:2 and 1:3,both the slope of the standard curve and the intensity of the signal decreased.In the study of the effect of p H,we found that in lower p H HRP was more effective when catalyzing ABTS to generate blue-green ABTS·+ radicals.Under the experimental conditions optimized based on UV-vis experiments,we constructed the standard curve of the ESR signal vs.the concentration of rabbit Ig G.After the background signal was substracted,there was no significant difference whether using double-integral area or peak-to-peak amplitude as the ESR signal.We found from this work that ESR could be used as the detector for ELISA.Fruits are rich in antioxidants such as vitamin C,polyphenols,and carotenoids.Since these antioxidants can reduce Cu2+ ion,we used both ESR and UV-vis methods to measure the antioxidant capacity of fruits.Vitamin C equivalent antioxidant capacity(VCEAC)was used to quantify the antioxidant capacity.The standard curve of the antioxidant capacity vs.the concentration of vitamin C was constructed.VCEAC values of the fruit samples were derived from the standard curve.The VCEAC values obtained by the ESR and UV-vis measurements are highly correlated with the correlation coefficient equal to 0.99.The VCEAC values determined by the ESR method are also highly correlated with those determined by the traditional 1,1-diphenyl-2-picrylhydrazyl(DPPH)method.