Capillary Electrophoresis With Capacitively Coupled Contactless Conductivity Detection For Biochemical Analysis

Capillary electrophoresis（CE）is a nanoscale liquid-phase separation technique that uses a capillary as the separation channel and a high-voltage DC electric field as the driving force to achieve the separation of analytes based on the mobility and partition behavior of sample components.CE has the advantages of short analysis time,high separation efficiency,multiple separation modes,and low sample and reagent consumption,and is particularly suitable for the analysis of ionizable substances.Capacitively coupled contactless conductivity detection（C~4D）is a general-purpose electrochemical detection technique based on the conductivity difference between the target analytes and the background electrolyte,theoretically responding to all charged substances.The C~4D detector has a simple structure and is convenient to couple with CE for on-column nondestructive detection,making it an ideal detector for CE.Nowadays,capillary electrophoresis with capacitively coupled contactless conductivity detection（CE-C~4D）has been widely used in various fields such as food,pharmaceutical,environmental and biochemical analysis.Based on the laboratory-built CE-C~4D system,the following three studies on biochemical analysis were carried out in this dissertation:A method for the analysis of endogenousγ-hydroxybutyric acid（GHB）in human urine and exogenousγ-hydroxybutyric acid in spiked beverages was established employing Good’s buffers as the background electrolytes;An ultrasensitive analytical method for acetylcholine（ACh）was developed based on the field-amplified sample injection（FASI）on-line preconcentration technique,and the method was applied to the detection of ACh in human cerebrospinal fluid;Ultrafiltration centrifuge tube was used as the reaction vessel for acetylcholinesterase（AChE）enzymatic reaction,and the substrate and product were detected by CE-C~4D.The developed method was applied to the activity determination,enzymatic reaction kinetics study,and inhibitor screening of AChE.This dissertation is divided into four chapters:Chapter One:Firstly,capillary electrophoresis was briefly introduced,including its development history,separation modes,application range,and detection techniques.Then,starting from the CE detection methods,the capacitively coupled contactless conductivity detection was introduced,and its development history and detection principle were described in detail.Finally,a comprehensive summary of the applications of CE-C~4D in food,pharmaceutical,environmental,and biochemical analysis in recent years was presented.Chapter Two:A simple and efficient CE-C~4D method was developed for the analysis of GHB employing Good’s buffers as the background electrolytes based on the laboratory-built CE-C~4D system.Through theoretical discussion and experimental optimization,the separation of GHB and related positional isomersα-hydroxybutyric acid andβ-hydroxybutyric acid was achieved within 4 min using 150 mM 4-（2-hydroxyethyl）-1-piperazineethanesulfonic acid（HEPES）as the running buffer.Under the optimized condition,the relative standard deviations of migration time and peak area were less than 1.1%and 4.5%,indicating good precision.The C~4D signal of GHB showed a good linear relationship with GHB concentration in the range of 3-300μM with a determination coefficient of 0.9997,and the detection limit was calculated to be0.37μM based on the signal-to-noise ratio of 3.Furthermore,liquid-liquid extraction and solid-phase extraction（SPE）were comparatively studied for sample matrix purification.Combined with the optimized SPE procedure,the developed CE-C~4D method has been successfully applied for the determination of exogenous GHB in spiked beverages and endogenous GHB in human urine.Chapter Three:Based on the above CE-C~4D system,a conventional CE-C~4D method using the hydrodynamic sample injection approach was developed for the analysis of ACh and its structural analog choline（Ch）.On this basis,FASI was used to improve sensitivity to achieve the ultrasensitive detection of ACh.Through systematic optimization of electrophoretic conditions and FASI conditions,10 mM acetic acid was selected as the background electrolyte,and the sample was prepared in acetonitrile/water（90/10,v/v）.After introducing a section of water plug into the inlet end of the capillary,the sample was subsequently injected by an electrokinetic method at 10 kV for 8 s,and the rapid separation of ACh and Ch was achieved within 110 s at a separation voltage of+20 kV.The method showed good linearity and precision,with the detection limit as low as 0.058 nM for ACh,achieving up to 1054-fold enrichment compared to common hydrodynamic injection.Further combined with the sample processing technique of weak cation exchange SPE,the developed method has been applied to the determination of trace amounts of ACh in human cerebrospinal fluid.Chapter Four:The use of ultrafiltration centrifuge tube as the reaction vessel for AChE enzymatic reaction combines the advantages of relatively high affinity between the free enzyme and the substrate and the reusability of the immobilized enzyme to a certain extent.And the conventional CE-C~4D method established in Chapter three was used directly for the analysis of the natural substrate ACh and its hydrolysis product Ch in the enzymatic reaction solution.Under the optimal incubation conditions,the activity of AChE was determined using the developed method,and the peak area of Ch showed a good linear relationship with AChE concentration in the range of 0.1-20 mU/mL with a low detection limit of 0.018 mU/mL.The enzymatic reaction kinetics of AChE was also investigated,and the₈)of AChE was measured to be 0.24 mM in experimental conditions.In addition,tacrine（Tac）and huperzine A（Hup-A）were selected as the model inhibitors to evaluate the feasibility of the developed method for AChE inhibitor screening.The half-maximal inhibitory concentrations obtained from the inhibition curve were 0.035μM and 0.40μM for Tac and Hup-A,respectively.The above results demonstrate the applicability of the developed method in the field of enzyme assay and it is expected to be further applied to the determination of AChE activity in human body fluid samples and the screening of AChE inhibitors in traditional Chinese medicine.