| Osteosarcoma is the most common malignant bone tumor and occurs mostfrequently during adolescence. Due to its high malignancy degree, poor prognosis andthe metastasis happened in early stage, the osteosarcoma has unfavorable effect on thephysical and mental health of adolescents. In recent years, with the development ofneoadjuvant chemotherapy and radical surgery, the five-year survival rate ofosteosarcoma increases to60%70%. However, due to the multidrug resistance oftumor cell to chemotherapy, there are many patients with failure treatment because ofrelapse and metastasis of tumor.The drug resistance of osteosarcoma is correlated with the drug resistanceprotein expressed by the corresponding drug resistance gene, the detection ofosteosarcoma drug resistance protein plays an important role in pathological gradingof osteosarcom, drug monitoring during chemotherapy, assessment of curative effect,screening suitable anti-tumor drug for patients, also, it could provide scientificexperiment basis for individual chemotherapy and prognosis in clinical practice.In recent years, the monitoring methods of drug resistance protein includedELISA, immunohistochemistry, gene chip and RT-PCR, etc. But there were somelimitations in these methods, such as low sensibility, complex operation, lengthenedanalysis times, higher cost and so on. It is urgent to contrive a analysis methodincluding shortened analysis time, simple, sensibility and economic. Electrochemicalimmunosensor and chemiluminescence enzyme immunoassay provide a new way tosolve this difficult problem.We design and establish two electrochemical immunosensor analysis methodsand a chemiluminescence enzyme immunoassay method which succeed to detect multidrug resistance related protein of osteosarcoma. It is hopeful to real time monitormultidrug resistance between of chemotherapy of osteosarcoma, adjustchemotherapy scheme timely, realize individual chemotherapy, and enormouslyimprove prognosis of patients. It consists of three chapters:Chapter1Label-free electrochemical immunosensor based on K3[Fe（CN）6] assignal for facile and sensitive determination of tumor necrosis factor-alphaUsing K3[Fe（CN）6] as the label-free signal material, buiding the electrochemicalimmunosensor which was modified with nafion/K3[Fe（CN）6]-chitosan-glutaraldehyde（K-CS-GA） in this section. According to the antigen-antibody reactionprinciple, the immunosensor was used to accurately detect the tumor necrosisfactor-alpha （TNF-α）. The different modified electrodes were characterized viascanning electron microscopy （SEM）, cyclic voltammetry （CV） and invertedflurescence microscopy. The prepared K-CS-GA solution was characterized byultraviolet and visible spectrophotometry （UV-VIS） and infrared spectrophotometry（FTIR）. In addition, all experimental conditions were optimized in the wholeexperiment. The approach show that the immunosensor had high sensitivity, goodstability and low cost. Under optimal experimental conditions，the linear rangecovered from0.02to34ng mL-1, the detection limit had been as10pg mL-1. Theresult of detecting osteosarcoma serum sample was satisfactory. Thus, theimmunosensor would be promising in clinical application.Chapter2Sensitive electrochemical immunoassay of Metallothionein-3basedon K3[Fe（CN）6] as redox active signal and C-dots/nafion film for antibodyimmobilizationBuilding a C-dots/nafion and K-CS-GA solution modified electrochemicalimmunosensor by dropping-coating way in this section, was used to detect themetallothionein-3（MT-3）, which was based on immunoreaction. The K3[Fe（CN）6]was used as the electrochemical redox active material, and the nanomaterial C-dotswas used to amplified the anodic peak signal of differential pulse volt-ampere curve.Firstly, the K-CS-GA solution and C-dots/nafion compound were successively modified the prepared GCE. Secondly, the capture antibody assembled theC-dots/nafion compound film of the modified electrode. Then, the current signal ofdifferential pulse volt-ampere changed when the antigen-antibody reaction happened.The experimental result show the immunosensor possessed good specificity under theoptimal experimental conditions. The linear range of detection covered from5pg mL-1to20ng mL-1, the detection limit had been up to2pg mL-1. The detectionlimit of proposed method was obviously lower than the other detection method ofmetallothionein-3（MT-3）. The result of detecting osteosarcoma serum wassatisfactory, if it was compared with the result of enzyme-linked immuno sorbentassay （ELISA）.Chapter3Microplate chemiluminescence enzyme immunoassay for thedetermination of tumor necrosis factor-alpha in human serumA micro-plate based chemiluminescent immunoassay for measurement of TNF-α in human serum was built. The method had high sensitivity, high specificity,rapidity, and reproducibility. Experimental conditions, such as temperature, coatingbuffer, the volume of substrate, incubation time, dilution ratio and other relevantvariables upon the immunoassay had been examined and optimized. The proposedmethod exhibited high performance which the linear range was10-2000pg ml-1andthe detection limit was5.46pg ml-1. A coefficient of variance of less than10%wasobtained for both intra-assay and inter-assay precision. The recovery was between90-110%,The present method had been successfully applied to the analysis of TNFαin human serum. Good correlations were obtained between the results by the proposedmethod and the commercial radioimmunoassay kit. The proposed method exhibitedgood potential in the fabrication of TNF-αdiagnostic kits which could be used in theclinical analysis. |
