Preparation Of Functionalized Graphite Nanosheets And Their Application In The Preparation Of Electrochemical Immunosensors

The incidence of cancer worldwide has shown an increasing trend year by year,and detection of tumor markers is one of the effective ways for early screening and detection of asymptomatic micro tumor.Prostate specific antigen(PSA),as an intracellular glycoprotein produced only by the prostate,is an ideal tumor marker for clinical screening and diagnosis of prostate cancer.With the development of analytical technology,PSA detection method based on immunochemistry has been widely used.Due to the high stability of immune complexes and the sensitivity and selectivity of electrochemical methods for PSA detection,electrochemical immunosensors have become an effective tool for the detection of PSA.Printed carbon electrodes(SPCEs)are widely used in small electrochemical affinity biosensors due to their advantages of low cost,rapid preparation,no pretreatment and mass production.In the design and preparation of immunosensor,the core technology is the immobilization of biomolecules.Using different physical or chemical strategies to modify the surface of SPCEs for further immobilization of biomolecules has become an extensive research topic for researchers.At present,substances with different properties are usually used to modify the surface of SPCEs to functionalize SPCEs and then immobilize biomolecules.Although this method can be used for different types of biosensors,it has some disadvantages such as complex modification process,long time-consuming and inability for batch modification.In this study,firstly,graphite nanosheets with high electron transfer performance and good biocompatibility were prepared by ultrasonic peeling method,and were functionalized with 1-pyrenebutyric acid and polyethyleneimine to obtain PBA@GNSs and PEI @PBA@CNSs complexes.Then.the PBA@GNSs and PEI@PBA@CNSs complexes were modified on the surface of SPCEs by using the viscosity of the ink,and the antibody was covalently bonded to SPCEs through the carboxyl group of PBA and the rich amino group of PEI.This method is simple,rapid and can be used to modify SPCEs in batches.Based on the unique properties of PBA@GNSs and PEI@PBA@CNSs complexes,the functional biosensor active interface was constructed,which greatly improved the analyze performance of the immunosensor.Finally,the Coulomb method was used to measure the background electricity and the total electricity at the same time,and the difference between them was used as the electrical signal to quantify the PSA content.The constructed two sandwich electrochemical coulometric immunosensors successfully realized the accurate determination of PSA.The main content of this thesis is as the following three chapters:Chapter 1 IntroductionIn this chapter,firstly,the research background of PSA was briefly summarized,and the current detection methods and technology status of PSA were introduced.Secondly,the electrochemical immunosensor was introduced in detail,including its basic principle,classification,design strategy,immobilization of biomolecules on the surface of SPCEs and its application in PSA detection.Then,the graphite nanosheets and their preparation methods were summarized,followed by a brief overview of the application research of pyrene derivatives and polyethyleneimine in electrochemical immunosensors.Finally,the background and research content of this thesis were clarified.Chapter 2 Studies on PSA detection using printed carbon electrodes modified with exfoliated graphite nanosheets-pyrene nanostructureThis chapter is based on the printed carbon electrode which was modified by 1pyrenebutyric acid@graphite nanosheet(PBA@GNSs)composite synthesized in one step,to construct a disposable coulometric enzyme immunosensor for rapid detection of PSA.First,PBA non covalently functionalized graphite nanosheet composites(PBA@GNSs)were prepared using the method of ultrasonication exfoliation with PBA as the surfactant.Then,the PBA@GNSs composite was spread on the surface of the freshly brushed printed carbon electrode to achieve batch modification.Next,Ab1 was immobilized on the electrode by the occurrence of condensation reaction between carboxyl group and amino group to form amide bond,and PSA antigen as well as HRP labeled secondary antibody was bound to the electrode surface by immunospecific recognition to form sandwich immune complex.Finally,the substrate H2O2 was added.In the process of catalyzing the decomposition of H2O2,HRP formed an electrochemical oxidation cycle with the electron mediator PVI-Os electrodeposition on the immune complex.In the end,Os3+ gained electrons on the electrode surface to generate a reduction power which had a linear relationship with the PSA concentration,and the detection of PSA can therefore be realized.Chapter 3 PEI@PBA@CNSs complex-based coulometric immunosensor for ultrasensitive detection of PSAIn this chapter,the printed carbon electrode modified by PEI@PBA@CNSs complex was used as the base electrode to prepare a cheap and highly sensitive electrochemical immunosensor for the study of PSA.First,the PEI@PBA@GNSs composite was obtained by covalent bonding of PEI and the previously synthesized PBA@GNSs complex.As the polymer with the highest amino group density,PEI can provide more antibody binding sites for the immunosensor,and greatly increase the load and electrochemical signal of the captured antibody.Then,the PEI@PBA@GNSs complex was spread on the surface of SPCEs to achieve batch modification.After that,the PSA antibody was crosslinked on the surface of the modified electrode with glutaraldehyde as the crosslinking agent.The sandwich detection mode was used.After the sandwich complex was formed on the electrode surface,a layer of redox macromolecules was deposited on the immunocomplex as the electron mediator of the marker enzyme HRP using the method of electrodeposition,and finally coulombic detection was performed.The difference between total and background charge(Ct-2Cb)showed good linear relationship with PSA concentration in the range of 0.2-100 pg/mL,and the detection limit was as low as 0.067 pg/mL(S/N=3),so the ultra sensitive analysis of PSA was successfully achieved.