| There are certain difficulties to make fast and sensitive detection of complex samples in the field of pharmaceutical analysis.In recent years,the electrochemical sensor technology in the field of electrochemical analysis has shown significant advantages when it was applied to the rapid and sensitive detection of complex samples,especially being applied in the analysis of disease markers and Chinese herbal medicinal ingredients.Rheumatoid arthritis?RA?can cause permanent damage to joints,which is harmful to the human body.Early diagnosis is great significance for the treatment of RA patients.Moreover,puerarin is the main active ingredient in traditional Chinese medicine pueraria,and is the main indicator of quality evaluation of traditional Chinese medicine pueraria.In order to achieve rapid and sensitive analysis of RA disease markers and puerarin,based on the protein characteristics of RA disease markers and the electrochemical activity of puerarin,this paper used the principles of electrochemistry,biochemistry and physical chemistry,adopted new electrode modification materials,optimal reaction conditions and detection system.Finally,electrochemical sensors for rapid and sensitive detection of anti-cyclic citrullinated peptide antibody,Connective tissue growth factor,and puerarin were constructed.The proposed method provides a new analysis method for the auxiliary diagnosis of clinical RA,and also provides a convenient method for the evaluation of the quality of Chinese medicine pueraria and the sensitive analysis of the electroactive component puerarin in actual samples.The research contents of this thesis mainly include the following aspects:1 Label-free electrochemical immunosensor for sensitive detection of anti-cyclic citrullinated peptide antibodiesAnti-cyclic citrullinated peptide antibody?anti-CCP?is one of the specific biomarkers of rheumatoid arthritis?RA?,whose sensitive detection is essential for early diagnosis of RA.In this study,a label-free electrochemical immunosensor for anti-CCP detection was constructed by the aid of the excellent electrochemical properties of nitrogen-doped graphene and the good biocompatibility of gold nanoparticles?AuNPs?,according to the principle that proteins can be immobilized onto the AuNPs through the electrostatic adsorption.The fabrication process of the immunosensor was investigated by square wave voltammetry?SWV?and electrochemical impedance spectroscopy.SWV was employed to monitor the binding events between CCP and anti-CCP under optimized sensor construction conditions and detection conditions.Since the formation of the antigen-antibody immune complex introduces a barrier to electron transfer between the medium and the electrode,the elevating concentration of anti-CCP leads to the reduction of the current response.The proposed non-enzyme and label-free immunosensor not only had simple construction process,but also exhibited high specificity,selectivity and stability in the linear calibration curve range from 0.1252000 pg mL-1,with the detection limit of 0.0125 pg mL-1.The immunoassay was also applied for analysis of human serum samples spiked with anti-CCP.Therefore,it is potential to design the portable immunosensors based on AuNPs and N-G nanocomposites for anti-CCP sensitive detection as well as future rheumatoid arthritis diagnostics.2 Electrochemical Immunosensor Based on Alkaline Activated Nitrogen Doped Graphene for Determination of Connective Tissue Growth FactorConnective tissue growth factor?CTGF?is a cysteine-rich mitotic peptide that is closely related to fibrosis of multiple tissues and organs.It can be used as a potential biomarker for the diagnosis of rheumatoid arthritis?RA?.Utilizing the principle that the amino group on the protein can form an amide with the carboxyl group on the surface of the carbon material to form a strong bond.In this work,alkali-activated nitrogen-doped graphene?aN-G?with a three-dimensional pore structure was used as the electrode base material,an electrochemical immunosensor for sensitive detection of CTGF was successfully constructed.The working principle of the label-free immunosensor is that the combination of CTGF and antibodies forms an insulating layer on the electrode surface that hinders electron transfer,which will cause the reduction of the current response signal.Scanning electron microscopy,infrared spectroscopy,Raman spectroscopy,and electrochemical cyclic voltammetry and impedance methods were used to characterize the surface morphology and performance of the modified electrode.Differential pulse voltammetry was used to construct a standard curve for CTGF detection with a linear range of 0.0625125 pg mL-1,1252000 pg mL-1;the detection limit is 0.0625 pg mL-1.In addition,the prepared sensor showed good selectivity and stability in the analysis of the spiked human serum samples.It can also accurately detect CTGF indicators in serum samples of diagnosed RA patients.The sensor provides a new method for the rapid detection of CTGF in complex samples,and is expected to become a new method for clinical diagnosis of RA.3 A Novel Electrochemical Sensor Based on Ultra-micro Activated Carbon for the Determination of PuerarinAfter intensely ultrasonic cracking,activated carbon obtained larger specific surface area in the premise of the high electric conductivity being maintained,which has provided a prerequisite for the construction of highly sensitive electrochemical sensors.An electrochemical sensor for sensitive analysis of puerarin in Radix Puerariae was successfully constructed using the prepared ultra-micro activated carbon in this work.The electrochemical behavior of puerarin on the sensor was investigated by cyclic voltammetry and differential pulse voltammetry.Moreover,the conditions of sensor preparing and puerarin detecting were optimized.Under the optimal conditions,the oxidation peak current increased linearly with the concentration of puerarin in the range of 2.4×10-7 mol L-11.54×10-55 mol L-1,and the detection limit was 6.4×10-8 mol L-1.In addition,the proposed sensor also exhibited good reproducibility,stability and selectivity.With superior performance in sample analysis,the proposed method would not only provide a new way for rapid and sensitive detection of puerarin,but also give a new idea for the application of activated carbon materials in the field of electrochemical sensors. |
PDF Full Text Request