Surface Functionalization Of Graphene Oxides And Its Applicationes In Development Of Electrochemical Immunosensors For Cytokines

Cytokines are small soluble proteins(-6-70 kDa)secreted by cells,and play a very important part in the body's immune system.Many studies have suggested that the majority of cytokines could serve as biomarkers of cancer.Therefore,the quantitative detection of cytokines is of great clinical significance.However,the content of cytokines in the body is low(pM range),and many cytokines form the complex cytokine network.Thus,there is an urgent need to develop sensitive and multiplex methods for the quantitative detection of cytokines.Electrochemical immunosensors have been widely studied in recent years because of the simple operation,accurate detection rate,and high sensitivity.In recent years,the introduction of nano-materials has greatly improved the sensitivity of the sensors,and the introduction of monoclonal antibody used as capture antibody to the sensing interface is helpful to increase the specificity of the sensors greatly.Thus electrochemical immunosensors have great potential for cytokine detection.In this paper,we firstly focus on the development and characterization of graphene oxide(GO)based nanomaterials.Then aryl diazonium salt chemistry is used for modification of electrodes with these prepared GO nanomaterials for the development of two electrochemical immune biosensors for the detection of cytokines TNF-? and IL-6.These sensors demonstrate good stability,high selectivity and sensitivity.Finally,the proof-of concept of an electrochemical immunosensors for the simultaneous detection of two cytokines was proposed and successfully verified.The contents of this paper are summarized as follows:(1)The preparation and characterization of GO based nanocomposites.(a)Aryldiazonium salt chemistry was used to modify 4-aminophenyl terminated GO through C-C covalent bonding to obtain GO-ph-NH2.Then NaBH4 and HAuCl4 were added to load AuNPs on GO-ph-NH2 to prepare GO/AuNP.It was chacracterised by High-resolution transmission electron microscopy(HRTEM)..UV-Vis?XRD and so on.(b)The signal molecules such as ferrocene(Fc)aniline or nile blue(NB)aniline were modified to GO by aryldiazonium salt chemistry and ?-? conjuction,and then tumor necrosis factor(TNF-?)polyclonal antibody or interleukin 6(IL-6)polyclonal antibody was modified on GO by amide bonding to obtain the signal report GO-ph-Fc or Ab2-GO-NB.GO-Fc was chacracterised by FT-IR,GO-NB was chacracterised by UV-Vis?FT-IR?XRD and Raman.(2)In this part,an electrochemical immunosensor based on GO was successfully developed for the quantitative detection of IL-6.GO was firstly modified on gold electrodes by aryldiazonium salt chemistry followed by the attachment of IL-6 capture molocolonal antibody.Finally,a sandwich assay was fabricated.The quantitative detection of IL-6 was achieved by observing the change of electrochemical signal from the signal report Ab2-GO-NB.(3)We developed an electrochemical immunosensor based on GO/AuNPs nanocomposites for the quantitative detection of TNF-?.GO/AuNPs was firstly modified on the gold surface by C-C covalent bond through aryldiaznium salt chemistry,and N2+BF4*-ph-COOH was then fixed on AuNPs by aryldiazonium salt chemistry for the immobilization of TNF-? capture monoclonal antibody.And finally a sandwitch assay was developed.The quantitative detection of TNF-? was achieved by observing the change in Fc electrochemical signal from the signal report Ab2-GO-ph-Fc.(4)Finally,we integrated the first three parts of the work to develop a proof-of-concept electrochemical immunosensor for the simultaneous detection of TNF-? and IL-6.The feasibility of this immunosensor was demonstrated successfully.