The Study Of Osteosarcoma Cell Imaging And Detection Of Tumor Markers Warning Osteosarcoma Micrometastases

Osteosarcoma is one of the most common malignant tumors of bone. Hematogenous metastasis of osteosarcoma occurs early with a high incidence rate. The metastasis even happens in the early stage of osteosarcoma. Most metastases towards the lung happen with some to brain, kidney and so on. Osteosarcoma metastasis, especially for the micro-metastasis, is one of the most important factors in patients with osteosarcoma survival. Therefore, it is urgent to find a new method for the diagnosis of osteosarcoma, in particular, it can predict the early micro-metastasis of osteosarcoma, and the clear diagnosis of micro-metastasis was applied to guide the diagnosis and treatment of osteosarcoma.Blood metastasis is the main kind of osteosarcoma metastasis. Unattached tumor cells reached and attached special target organ with the blood circulation. Identification and counting the circulating tumor cells of osteosarcoma in blood is important for the judgment and precaution of metastasis. However, a single monitoring of tumor cells may lack the sensitivity and specific, some blood biomarker especially for tumor biomarker should be combined with the cell monitoring to judge the osteosarcoma metastasis.A new kind diagnostic platform, cell imaging of combined with electrochemical immunosensors for tumor biomarkers was fabricated in this work. The imaging of two kinds of osteosarcoma cells and the corresponding tumor biomarkers of osteosarcoma of tumor necrosis factor-alfa(TNF-?) and CEA were obtained. The main effort of this work was to develop a useful testing technology for the clinical diagnosis of osteosarcoma metastasis. And then, more detailed inspection processes will be operated to the accurate diagnosis of osteosarcoma. Furthermore, the diagnosis can guide the setting of adjuvant chemotherapy and surgery program. The full text of the work is divided into three parts, as follows:Part 1. Experimental study on the establishment of osteosarcoma cell imaging system based on 7-OH coumarin- MnO2 compositesA "signal-on" fluorescence imaging system based on the adsorption of 7-OH coumarin on the surface of MnO2 nanosheets for the osteosarcoma cell imaging was developed in this section. 7-OH coumarin was used as the fluorescent probe, and MnO2 was used as the quenching platform. And then the fluorescence quenching system of 7-OH coumarin- MnO2 composites for the sensing and cell imaging was fabricated. Glutathione(GSH) can reduce MnO2 nanosheets to be Mn2+, and release the adsorbed 7-OH coumarin. The fluorescence was recovered due to the released 7-OH coumarin, thus the concentration of GSH can be detected through the recovered fluorescence intensity. With the optimized experiment conditions, the linear range of GSH is from 0.03?M to 400 ?M. Furthermore, the ideal result was obtained by this method for the sensing of glutathione tablets as the real sample. Satisfactory results were also obtained in the further application of this system in osteosarcoma cell imaging. Therefore, the fabrication of 7-OH coumarin-MnO2 composites for the detection of GSH system and the cell imaging is practical. The result of this chapter can lay the foundation of cell detection for the clinical diagnosis of osteosarcoma.Part 2. Experimental study of electrochemical immunosensor based on CNT/CS modified electrode for the detection of TNF-?.CNT/chitosan composites(CNT-CS) were applied to modify GCE through dropping method in this section. Later, with the covalent effect of glutaraldehyde, TNF-? antibody was immobilized on the surface of CNT-CS modified GCE. According to immune-reaction, sandwich electrochemical immunosensor was fabricated for the sensitive detection of TNF-? based on CNT-CS modified GCE. The preparation of the immunosensor was characterized through electrochemical impedance spectroscopy(EIS) and cyclic voltammograms(CV), and the experimental conditions were optimized. The prepared immunosensor is high sensitive to TNF-? in the concentration range of 0.005 ng/mL to 34 ng/mL, with a detection limit of 0.0025 ng/mL. The sensing results of TNF-? illustrates that the fabricated immunosensor has the characters of stability, sensitivity, selectivity.Part 3. Electrochemical immunoassay based on polythionine as signal source for detection of carcinoma embryonic antigenIn this work, we fabricated a simple and reliable amperometric immunosensor based on a nafion/ polythionine(nafion/PTh) modified GCE for the precise and sensitive detection of CEA. The advantages of prepared immunosensor are as follows: the proposed strategy provided a simple and convenient method to fabricate the immunosensor and had acceptable performance for the response for CEA; nafion with excellent biocompatibility was used to stabilize electrochemical signal and bound adequate amount of anti-CEA antibody on the modified electrode. The response of the prepared immunosensor had a good linear relation with CEA concentration from 1×10-12 to 1×10-6 g/mL with a 0.1 pg/mL of LOD. Meanwhile, the immunosensor was applied to determinate CEA levels in the human serum samples, the results of which were satisfactory, comparing with that of chemiluminescent immunoassay.