Study On Electrochemical Sensors For The Detection Of Homocysteine

Electrochemical sensor technology has the advantages of low cost,simple operation,portability,high sensitivity,and rapid response.It has gradually become one of the research hotspots in the fields of medical detection,pollutant detection,and food safety detection.In an electrochemical sensor,the recognition element plays a role of specific recognition and catalytic substrate,which determines the sensitivity and selectivity of the sensor.At present,there are many studies on the use of electrochemical sensor technology to detect important compounds in the human body.However,due to the complex composition of human body fluids and many interferences,the wide application of electrochemical sensor technology in the field of medical detection is limited.Therefore,under the premise of ensuring that the sensitivity and detection limit meet the actual detection requirements,the construction of an electrochemical sensor that has strong anti-interference ability and can be used in actual samples requires further exploration by scientific researchers.Homocysetine(Hcy) is an intermediate metabolite in the methionine cycle.At present,more and more studies have reported that abnormal levels of Hcy in the human body are related to a variety of diseases,and the higher the level of Hcy,the higher the risk of disease.Therefore,the detection of Hcy is of great significance for clinical treatment and health assessment.In this thesis,nanoporous gold(NPG),which has both nanostructure and catalytic function,is used as the recognition element to modify the glassy carbon electrode(GCE),and the nanoporous gold/glassy carbon electrode(NPG/GCE)is constructed.Through scanning electron microscope(SEM),it is observed that NPG has nano-scale pore size and ligaments.Using the constructed NPG/GCE electrode to detect Hcy,it is found that the NPG/GCE electrode has better catalytic activity for Hcy than the GCE electrode,and the Hcy on the surface of the NPG/GCE electrode is an irreversible surface-controlled oxidation reaction.In the detection range of 3～100 μM,the current response of the NPG/GCE electrode has a good linear relationship with the concentration of Hcy.The sensitivity is 36.75 μA mM-1 cm-2,and the detection limit is as low as 0.99 μM,which can meet the needs of actual detection.In addition,the constructed NPG/GCE electrode shows strong anti-interference ability in the presence of some common substances in human urine,and the detection is more accurate in the spike recovery experiment of diluted urine samples.However,the NPG/GCE electrode uses NPG as the recognition element,which has low specificity,not only can catalyze Hcy,but also Cys,and the peak potentials of Hcy and Cys are close,which makes it impossible to detect selectively in samples containing Hcy and Cys.Methionine γ-lyase(MGL)is a pyridoxal-5-phosphate(PLP)dependent enzyme that can catalyzes Hcy into hydrogen sulfide(H2S),ammonia and α-butanone acid.In order to improve the anti-interference ability of the Hcy electrochemical sensor,this thesis takes the mgl gene of Trichomonas vaginalis as the target gene,and obtains MGL that specifically catalyzes Hcy through prokaryotic heterologous expression.Then,the MGL protein that specifically catalyzes Hcy is used as the biological recognition element,and the NPG with good biocompatibility and high catalytic activity is used as the enzyme immobilization carrier to construct the MGL/NPG/GCE biological enzyme electrode.The constructed MGL/NPG/GCE electrode realizes the detection of Hcy through the synergistic catalysis of MGL and NPG.The MGL/NPG/GCE electrode exhibits a good linear response in the detection concentration range of Hcy from 5 to 100μM,the sensitivity is 40.10 μA mM-1 cm-2,the detection limit is as low as 1.24 μM,and it has good reproducibility.In addition,the constructed MGL/NPG/GCE electrode exhibits a good anti-interference ability to common substances in urine,and the interference of Cys to Hcy detection is controlled at about 20%.Cystathionine β-synthase(CBS) is a PLP-dependent enzyme in the methionine cycle that can catalyze three reactions to produce H2S.Among them,CBS catalyzes the reaction of Cys and Hcy to produce Cystathionine and H2S,which is the main way to produce H2S.The amount of H2S produced by the remaining two reaction pathways is negligible.In order to reduce the interference of Cys on the detection of Hcy,this thesis uses cbs in Pseudomonas aeruginosa PAO1 as the target gene to express the CBS protein heterologously in the prokaryotic system.HPLC detection results showed that when Cys alone exists,CBS protein does not catalyze Cys,which can eliminate the interference of Cys on the detection of Hcy.CBS/NPG/GCE electrodes were constructed based on the CBS protein that specifically recognizes Hcy as the biological recognition element,and NPG with good structure and function as the enzyme immobilization carrier.The CBS protein in the CBS/NPG/GCE electrode uses Hcy and Cys as substrates to generate H2S,and then NPG catalyzes H2S to generate sulfur and polysulfides to generate an oxidation current.In the concentration range of 5～100 μM,the CBS/NPG/GCE electrode has a good linear response to Hcy,the sensitivity is 10.44μA mM-1 cm-2,the detection limit is as low as 1.31 μM,and the relative standard deviation(RSD)is less than 3.29%.In particular,the constructed CBS/NPG/GCE electrode uses CBS as the identification element,and the synergy between CBS and NPG not only realizes the sensitivity of Hcy detection,but also solves the problem of Cys interference in Hcy detection.In this thesis,a non-enzymatic and biological enzymatic electrochemical sensor based on nanomaterials was constructed for the detection of Hcy.The combination of NPG with good biocompatibility and high-efficiency catalytic activity and specific biological enzymes is used to construct an electrochemical sensor for Hcy detection,which realizes simple,efficient,specific,sensitive and reliable rapid detection of Hcy.In addition,the Hcy sensor constructed in this paper has the advantages of easy preparation,rapid detection,and low cost,and has great practical application potential in clinical treatment and health assessment.