Study On Determination Of Immunoglobulin And Antioxidant Capacity By Electron Spin Resonance Based On Metal Probe

Electron spin resonance(ESR)spectroscopy,as a technology for detecting one or more unpaired electrons in compounds,has been developed and applied widely in various research fields over the past few decades.As only paramagnetic materials can be detected for ESR signal,the interference and background signals received during the measurement are very small.Therefore,ESR has some certain advantages in quantitative detection.In this dissertation,ESR technique was applied to immunoassay and quantitative detection of antioxidant capacity of fruits.Firstly,a novel ESR spectroscopy method for immunoassay was presented using iron oxide nanoparticles(FeNPs)as probe and rabbit immunoglobulin G(IgG)as the model antigen.Secondly,using copper sulfide(CuS)nanoparticles as the probe,the biotin-streptavidin system was introduced to develop an ESR immunoassay method with better sensitivity.Finally,based on the ESR signal of Cu²⁺,a method was developed to study the antioxidant capacity of various kinds of fruits.Antioxidant capacity of 22 kinds of fruits was measured by the developed ESR method.With 180 nm FeNPs as probes,a new ESR spectroscopy method based on the ESR signal of FeNPs was presented and applied to the determination of rabbit IgG.Goat anti-rabbit IgG polyclonal antibody was conjugated to FeNPs.The sepharose beads were used as solid phase and used to separate the analyte from the sample solution.Two typical immunoassay formats,sandwich and competitive formats,were applied to the determination of rabbit IgG.After the optimization of the assay,a molar ratio 500:1 of antibody to FeNPs was selected.The standard curves obtained with both sandwich and competitive formats fit well with the 4-parameter logistic model(4PLM).The limits of detection(LOD)obtained by sandwich and competitive formats were 0.13?g/m L and 0.56?g/m L,respectively.The results show that the LOD obtained by the sandwich format is better than that obtained by the competitive format.The two formats were applied to the determination of the rabbit IgG in rabbit serum.The diluted serum sample was directly analyzed and additional sample treatment was not required.The analytical results of the spiked samples indicated that the recoveries of the analytes were acceptable.Furthermore,the sample preparation time used in this method is shorter than that used in most existing immunoassay methods.Water soluble copper sulfide(CuS)nanoparticles were synthesized using L-cysteine as the ligand.The Cu²⁺ions enclosed in the nanoparticles were used as the probe to develop a novel ESR immunoassay method.Multiple biotins were conjugated to the antibody of rabbit IgG,and the streptavidin was attached to the CuS nanoparticles.The high affinity between biotin and streptavidin is taken advantage of in order to improve the sensitivity for the analyte's detection.After the immunoassay reaction was performed,large amount of Cu²⁺/Cu⁺ions inside the nanoparticles were released with the help of diethyldithiocarbamate(DDC)and the Cu²⁺-DDC complex formed.The Cu²⁺-DDC complex was extracted into n-butanol,which was used as the analytical sample.Both ESR and UV-vis signals were collected for the analytical sample.The LODs of the rabbit IgG obtained by ESR and UV-vis method were 1.76pg/m L and 2.36 pg/m L,respectively.The detection range using ESR as the detection method was from 8.8 pg/m L to 500 ng/m L,covering almost 5 magnitude orders.The rabbit serum was analyzed and the rabbit IgG concentration was found to be 7.76mg/m L.The reproducibility of the present method was good enough with the intra-assay relative standard deviation(RSD)within 3.4%and the inter-assay RSD within 11.2%.And the spiked serum samples were analyzed and the experimental results indicated that the recoveries were from 108.2 to 113.7%.The materials used to synthesize CuS nanoparticles are cheap and easily available.The synthesis conditions are mild,and the synthesis method is relatively simple,time-saving and environmentally friendly.Cu²⁺is released from the nanoparticles and extracted into the organic phase,which can effectively avoid the interferences from the sample matrix.There is no ESR signal in most biological materials so that the background signal does not exist.Compared with traditional enzyme-linked immunosorbent assay(ELISA)method,ESR spectroscopy which detects paramagnetic metal ions(Cu²⁺)would not be interfered with the color of the analytical sample.The analytical samples are also very stable and insensitive to heat and light so that the results of the analytical samples within at least 2 weeks are repeatable.An ESR method was developed for detecting the antioxidant capacity of fruits based on Cu²⁺sensor,and it was applied to measure the antioxidant capacity of 22kinds of fruits.Cu²⁺is reduced to Cu⁺by the antioxidants in the fruits,and the remaining Cu²⁺was detected to quantify the antioxidant capacity of various fruits.The results were shown as vitamin C equivalent antioxidant capacity(VCEAC)which was used to compare the antioxidant captivity of different fruits.The analytical sample can be determined by both ESR and UV-vis spectrometers.The VCEAC values obtained by ESR and UV-vis methods ranged from 24.23 to 688.61 mg/100 g and from 24.14 to 677.79 mg/100 g,respectively.Two traditional methods,1,1-diphenyl-2-picryl-hydrazyl free radical scavenging(DPPH)methods and cupric ion reducing antioxidant capacity(CUPRAC)were employed for comparison.Based on Pearson's correlation test,the results obtained by CUPRAC and DPPH methods were both significantly correlated with these obtained by the present method,which illustrated practical application of the novel method and indicated that it was reliable.Besides,total phenolic content for all kinds of fruits was measured with the Folin–Ciocalteu reagent,and VCEAC values obtained by the ESR method were significantly correlated with total phenolic contents.The resonant signal of Cu²⁺was measured and color from fruit samples will not affect the measurement results.However,for DPPH and CUPRAC methods,the colored analytical solution could influence the absorbance.In addition,both frozen fruit samples and measurement samples can be stored for a long time.The reagents used in this work are cheap,stable,and insensitive to light and heat.Cu²⁺was isolated from Cu⁺and sample matrix,as can effectively avoid the effect of complicated matrices and the oxidation of Cu⁺ to Cu²⁺.