Construction Of Graphene Three-dimensional Macroporous Electrode And Its Sensing Of Circulating Tumor Cells

Circulating tumor cells（CTCs）are closely related to cancer metastasis,so the detection of circulating tumor cells is of great significance for the disease analysis and treatment monitoring of cancer patients.In recent years,biosensors have been widely used in the detection of circulating tumor cells,and are gradually developing in the direction of miniaturization,low cost,high selectivity and strong sensitivity.In the process of biosensor construction,it is very important to select the appropriate material to construct the biological substrate.Graphene has become one of the important materials for building biosensors due to its excellent properties such as good chemical stability,electrical conductivity,large specific surface area and biocompatibility.At the same time,the combination of graphene and metal oxide nanomaterials has been well applied in the construction of new biosensors.Zinc oxide（Zn O）nanorods,as an important metal oxide nanomaterial,can not only increase the surface area of graphene composite nanomaterial,but also facilitate the surface modification of the substrate due to the large number of hydroxyl groups on the surface of Zn O nanorods.Therefore,it is an effective strategy to grow inorganic semiconductor material Zn O nanorods on the substrate of three-dimensional（3D）graphene to construct a highly sensitive and specific cell detection substrate.In this paper,through the combination of three-dimensional graphene and Zn O nanorods,the detection base of CTCs biosensor was successfully constructed,and the fabrication of3D macroporous structure electrode was completed,which realized the specific detection and sensing research of CTCs.The main research contents are as follows:1.A 3D graphene macroporous foam substrate with Zn O nanorod array was developed for specific and non-invasive detection of CTCs.First,using natural graphite as a raw material,we prepared a 3D graphene structure based on the template method.We used a two-step oxidation method to prepare graphene oxide（GO）based on natural graphite,and then let GO adhere to the surface of the nickel（Ni）template.After GO was dried,GO was reduced to reduced graphene oxide（r GO）with hydrazine monohydrate,and metal Ni was removed with a mixed solution of dilute hydrochloric acid and ferric chloride.The process of graphene oxide obtained by the two-step method and then reduced to r GO can make r GO have a higher conductivity,which is conducive to the subsequent detection of CTCs.Then we grow Zn O nanorods on the surface of the 3D graphene structure.We synthesized Zn O nanoparticles as seeds by Pacholski method.After the seeds were evenly distributed on the surface of 3D graphene structure,Zn O nanorods were grown on the surface of r GO by seed growth method to complete the preparation of r GO-Zn O foam structure.During the preparation of the three-dimensional graphene structure and the growth of Zn O nanorods,the substrate was characterized by a manual emission scanning electron microscope（SEM）.We found that the graphene foam can still exhibit a good macroporous self-supporting structure after removing the metal Ni,and after the Zn O nanorod array is closely and uniformly modified on the graphene surface,the r GO foam still maintains the macroporous structure.Raman spectra of GO foam,r GO foam and r GO-Zn O foam demonstrated that GO was reduced to r GO and Zn O nanorods were successfully grown on graphene substrates.The x-ray powder diffraction（XRD）patterns of r GO foam and r GO-Zn O foam also proved the successful growth of Zn O nanorods on graphene substrates.Finally,we performed biological modification on the r GO-Zn O foam structure to complete the binding of epithelial cell adhesion molecule antibody（anti-Ep CAM）to the substrate.With the help of the epithelial cell adhesion molecule antibody（anti-Ep CAM）and the array structure,the Ep CAM-positive CTCs can adhere on the detection substrate（r GO-Zn O-anti Ep CAM foam）.Finally,we demonstrated that CTCs were successfully captured by r GO-Zn O-anti Ep CAM foam substrate by SEM and fluorescence microscope photographs.2.While using the 3D graphene macroporous foam substrate to successfully capture CTCs,we have completed the specific and non-invasive detection of CTCs.We used a semiconductor testing system（Keithley 2601B）to detect the resistance change of the r GO-Zn O-anti Ep CAM foam substrate during the process of capturing CTCs,and used an electrochemical workstation（CHI 660D）to measure the electrochemical impedance spectroscopy（EIS）of the MCF-7 binding process.Compared to the Ep CAM-negative cancer cell,the resistance of the substrate is rising with the amount of the Ep CAM-positive CTCs.The possible reason is that the negative charges of CTCs affected the conductivity of the p-type reduced graphene oxide（r GO）,resulting in the increased resistance of r GO-Zn O-anti Ep CAM foam,when the CTCs binding to the r GO-Zn O-anti Ep CAM foam.In addition,the diameter of the semicircular curve of the EIS gradually increased as the cell was captured by the substrate.The increase in the diameter of the semicircular curve of the EIS indicated that the charge transfer resistance(R_ct)in the system increased,and the R（Q（RW））（CR）circuit was fitted by the Zsim Demo software to give the resistance value of R_ct,which more intuitively reflected the growth trend of R_ct.The increase of R_ct in the detection system may be due to the fixation of negatively charged MCF-7 cells on the substrate,which impeded the charge transfer process between the substrate and the solution.These results reveal that CTCs can not only be specifically captured by the r GO-Zn O-anti Ep CAM foam,but also the resistance of the detection substrate and the R_ct between the substrate and the solution were increased during the CTCs captured process.In conclusion,the construction of the detection substrate,the measurement and analysis methods of the electrical signals are of great significance in CTCs acquisition and sensing,which will provide an effective strategy for the real-time monitoring of CTCs at the clinic.