Establishment Of The Electrochemical Methods For Plasma Indole And Their Applications

Objective:1.To construct a new electrochemical method based on the signal amplification strategy by gold magnetic nanocomposite（Au/Fe₃O₄）for the sensitive detection of indole in plasma matrix.2.To construct a fast and sensitive electrochemical sensor for the detection of plasma indole based on the nitrogen-doped ordered mesoporous carbon（NOMC）-chitosan（CS）modified screen-printed carbon electrode（SPCE）.3.To detect the plasma indole in polycystic ovary syndrome（PCOS）patients and healthy women at childbearing age by the electrochemical sensor based on NOMC-CS/SPCE.And then to explore the effects of changes in indole concentration in physiological and pathological states,respectively.Method:1.Au/Fe₃O₄ was successfully synthesized by the irradiation of high-energy electron beam.Firstly,4-aminothiophenol（4-ATP）reacted with Au/Fe₃O₄ by Au-S bonds to form the product of Au/Fe₃O₄@ATP.Next,Au/Fe₃O₄@ATP was diazotized with NaNO₂ to generate a diazo compound.Finally,the diazo compound reacted with indole and produced Au/Fe₃O₄@ATP-azo-indole by the coupling reaction.The product was dropped onto the surface of SPCE and dried at room temperature,and then the SPCE was connected to the electrochemical workstation,with a magnet placed under the SPCE to gather the product.The product was determined by the differential pulse voltammetry（DPV）technique after dropping the supporting electrolyte solution onto the surface of SPCE.2.NOMC was added into the chitosan solution and ultrasonicated to prepare a black uniform NOMC-CS dispersion.And then the suspension was modified onto the working electrode of SPCE,and dried at room temperature.The plasma samples were pretreated by liquid-liquid extraction with ether.The extracted solution was spin-dried,and then the residual was redissolved in phosphate buffer saline（PBS）to form the working solution.Finally,the working solution was added onto the surface of SPCE for the detection of indole by DPV method.3.The electrochemical sensor based on NOMC-CS/SPCE was used to measure the concentrations of indole in plasma of 48 PCOS patients and 20healthy women at childbearing age,respectively.The clinical information and related laboratory test indicators of each subject were collected.And then the data were statistically analyzed to explore the relationship of the data and to investigate the clinical significance.Results:1.The linear range of indole measured by the electrochemical method based on the signal amplification strategy by Au/Fe₃O₄ was from 0.50 to120.00μg/L,and the regression equation wasΔI（μA）=0.0202 c（μg/L）+0.8121（R²=0.9923）.The limit of detection（LOD）was 0.10μg/L（S/N=3）.The relative standard deviations of intra-day and inter-day precisions were 4.9%and 8.2%,respectively.The recoveries were from 97.2%to105.4%.2.The electrochemical sensor based on NOMC-CS/SPCE was used to determine the concentrations of indole in human plasma.The regression equation of indole in the linear range from 1.0 to 150.0μg/L was y=0.0189 x+0.6652（R²=0.9962）.The regression equation of indole in the linear range from 1.0 to 20.0μg/L was y=0.0217 x+0.6442（R²=0.9975）.The LOD was 0.2μg/L（S/N=3）.The relative standard deviations of intra-day and inter-day precisions were 5.8%and 7.5%,respectively.The recoveries were from 95.0%to 103.2%.This method had a good correlation with high performance liquid chromatography-fluorescence detection（HPLC-FLD）for the determination of plasma indole（r=0.752,p<0.001）.3.The concentrations of indole in the plasma of PCOS patients and healthy women at childbearing age were determined by the electrochemical sensor based on NOMC-CS/SPCE,respectively.The levels of indole were statistically different between the two groups.The level of plasma indole in PCOS group was significantly higher than that in the healthy control group.Receiver operating characteristic（ROC）curve showed that indole had an excellent diagnostic efficacy for PCOS,which displayed the sensitivity was77.1%,the specificity was 95.0%,and the area under the curve（AUC）was0.904.Regression analysis showed that indole was a risk factor for PCOS（Odds ratio（OR）=4.848,p<0.001）.Furthermore,two subgroups according to the presence or absence of insulin resistance in PCOS patients were separated to form insulin resistance（IR）group and non-insulin resistance（Non-IR）group.The results showed that the physiological cycle,total bile acid（TBA）,some blood glucose detection indicators,all insulin detection indicators,homeostasis model assessment of insulin resistance（HOMA-IR）and homeostasis model assessment ofβ-cell function（HOMA-β）were statistically different in IR group and Non-IR group.Conclusions:1.A sensitive and cost-effective detection of indole in plasma matrix was achieved by the electrochemical method based on the signal amplification strategy by Au/Fe₃O₄,which provides a new tool for indole detection in conventional laboratories.2.An electrochemical sensor for the determination of indole in human plasma was constructed based on NOMC-CS/SPCE.This sensor had the advantages,including simplicity,quickness,good stability,repeatability and strong anti-interference abilities in indole detection.It provides a promising way for the detection of plasma indole in clinics.3.Disorders of indole levels were found in PCOS patients.The level of indole was found to have a correlation with insulin resistance in further analysis,suggesting that indole might have a potential correlation with the occurrence and development of PCOS.