Study On The Analysis Method Of Glycosylated Hemoglobin In Human Blood Samples By Capillary Electrophoresis And Contactless Conductivity Detection

In recent years,diabetes has increasingly endangered human health.Glycosylated hemoglobin（Hb A1c）is the"gold standard"for detecting long-term blood glucose levels in human blood,and its content is usually closely related to some complications.Therefore,it is often used to monitor blood glucose concentration to diagnose and treat diabetes.Internationally,most methods for detecting Hb A1c are expensive,time-consuming and labor-intensive.Therefore,in this paper,capillary electrophoresis（CE）-capacitively coupled contactless conductivity detection（C⁴D）was chosen to develop a simple,low-cost and accurate method for the determination of Hb A1c in human blood samples.C⁴D is a relatively novel detector connected to CE in recent years.It has the advantages of strong versatility,miniaturization and long-term lossless use.So far,there has been no research on the analysis method of hemoglobin content with CE-C⁴D,so the research results of this article will fill the technical gap in this part and provide research ideas and methods for the subsequent low-cost CE-C⁴D detection of other biological samples.Specifically include the following aspects:（1）In order to reduce the cost of research,we tried to use porcine hemoglobin instead of human hemoglobin to explore the response signal of hemoglobin with C⁴D detector.Firstly,the feasibility of replacing human hemoglobin with porcine hemoglobin was verified by CE-Ultraviolet（UV）method.Secondly,through the selection of the C⁴D detection system and the sample pretreatment experiments,it is preliminarily shown that 200 mmol/L acetic acid（HAc）+30μmol/L 1-methyl-3-dodecylimidazole bromide solution（p H=2.67）can obtain good response signals of porcine hemoglobin and human hemoglobin with C⁴D detector.However,in the subsequent separation experiments for Hb A1c,it is proved that the HAc system can provide a universal response signal,that is,the detection signal of C⁴D is not the characteristic response signal of hemoglobin at this time.Therefore,this system cannot be used for the separation of human hemoglobin and the determination of Hb A1c,but it provides a guiding method for the next attempt to detect human hemoglobin with C⁴D alkaline system.（2）A method for the determination of Hb A1c in human hemoglobin by CE–C⁴D was successfully developed.At the same time,the method is verified by UV detector in series.In this method,H₃BO₃ with low conductivity is selected as the background electrolyte（BGE）to reduce the background conductivity of BGE and other background interference,and the specific interaction between tetraborate and the sugar moiety of Hb A1c in the cis-diol mode is used to improve the separation of hemoglobins;the p H of the buffer is adjusted by the LiOH solution with low conductivity and can provide a stable baseline;the micromolar concentration of spermine is selected as the additive to reduce the influence of the additive on the conductivity of the system and the C⁴D detection background.The study also found that spermine can also inhibit the electroosmotic flow of system so as to improve the separation effect.After experimental optimization,the best experimental conditions for the detection of Hb A1c by CE-C⁴D were determined as follows:BGE was 100 mmol/L H₃BO₃+0.35 mmol/L spermine（p H was adjusted to 9.60 with 0.10 mmol/L LiOH）,the sample was injected at 20 mbar for 10 s,and the separation voltage was+15 k V.Under these conditions,the CE and C⁴D detectors can simultaneously separate three hemoglobin forms,and inject 6 needles in parallel,the intra-day and inter-day repetitions of migration time and peak area meet the requirements of analysis,and the linear correlation is good,which of all are greater than 0.990.Finally,the method was validated by ion exchange chromatography and CE-UV,and the results were not significantly different.（3）In order to obtain the best detection sensitivity of CE-C⁴D for detecting Hb A1c content in human hemoglobin,the sensitivity of hydrodynamic injection and electrokinetic injection for detecting hemoglobin was compared on the basis of the detection system of the second part.It was found that when the sample solution is 100 mmol/L H₃BO₃-LiOH（p H=9.60）,the detection sensitivity of electrokinetic injection is 11.1 times higher than that of hydrodynamic injection,which provides a clear solution for improving the sensitivity of sample detection.