基于直接电子传递型电化学免疫传感器和微孔板式化学发光免疫分析法快速检测骨肉瘤耐药相关蛋白的研究
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摘要
骨肉瘤是最常见的原发性恶性骨肿瘤，好发于青少年，恶性程度高，早期即可发生转移，预后差，是严重危害青少年身心健康的恶性肿瘤。近年来，随着新辅助化疗、手术等综合治疗的发展，骨肉瘤的五年生存率逐渐上升到60%~70%。但是，仍有不少的患者因肿瘤复发、转移而导致治疗失败，其主要原因是肿瘤细胞对化疗药物产生的多药耐药。
骨肉瘤耐药性与其耐药基因表达的标志性耐药蛋白密切相关，通过检测骨肉瘤标志性耐药蛋白，对骨肉瘤病理学分级、化疗过程中的药物监测、疗效评估、快速筛选适合患者的抗肿瘤药物有着极其重要的作用，并可为临床上实现个体化化疗和预后判断提供科学的实验依据。
目前用于骨肉瘤标志性耐药蛋白的临床监测方法主要包括酶联免疫吸附法（ELISA）、免疫组化染色法、基因芯片、RT-PCR等技术，但这些方法均存在一定的局限性，如敏感性不高、操作繁琐、周期长、费用高等问题，迫切需要开发研制一种快速、简便、灵敏、准确、经济的骨肉瘤耐药相关蛋白检测技术，电化学免疫传感器技术及化学发光免疫分析技术为解决这一难题提供了新的出路。
本文设计并研制了两种电化学免疫传感器和一种化学发光免疫分析技术，成功实现了骨肉瘤相关耐药蛋白（肿瘤坏死因子-α和金属硫蛋白-3）检测，为临床提供了新型的检测手段，有助于实现临床上骨肉瘤化疗过程中的实时药物耐药监测，以便及时调整化疗方案，实现骨肉瘤的个体化化疗，大大改善患者的预后。全文共分为三部分，分述如下：
第一部分：基于铁氰化钾作为指示信号的免标记电化学免疫传感器用于肿瘤坏死因子α(TNF-α)的超灵敏检测
本章节主要以K_3[Fe(CN)_6]作为免标记电化学信号物质，构建了nafion/K_3[Fe(CN)_6]-壳聚糖-戊二醛（K-CS-GA）复合物修饰的电化学免疫传感器，根据抗原-抗体反应原理，用于肿瘤坏死因子（TNF-α）的精密检测。利用扫描电子显微镜（SEM）、循环伏安技术（CV）和荧光倒置显微镜等对修饰电极的制备过程进行表征，以及利用紫外可见分光光度法（UV-VIS）和红外分光光度法（FTIR）对制备的修饰复合物K-CS-GA溶液进行表征。实验结果表明，在最佳实验条件下，该方法所制备的nafion-K-C-GCE免疫传感器具有较强的生物大分子识别能力，具有较高的灵敏度和较宽的检测范围，用于检测TNF-α的线性范围为0.02~34ng/ml，其检测限为10pg/ml，用于骨肉瘤血清样本测定结果令人满意，并且该免疫传感器合成方法简单易行，所用修饰复合物K-CS-GA溶液廉价易得并且具有长期稳定性。因此，该电化学免疫传感器具有广阔的临床应用前景。
第二部分：建立基于K_3[Fe(CN)_6]氧化还原信号和C-dots/nafion薄膜固定抗体的电化学免疫传感器用于金属硫蛋白(MT-3)的检测
本章节通过滴涂方式，构建了C-dots/nafion和K-CS-GA复合物修饰的电化学免疫传感器，以K_3[Fe(CN)_6]作为电化学氧化还原信号来源，通过应用C-dots对差分脉冲伏安曲线氧化峰电流信号进行放大，并且以免疫反应为基础，用于金属硫蛋白（MT-3）的精密测定。该方法先将电化学信号活性复合物K-CS-GA和C-dots/nafion复合物依次修饰在处理好的电极上，再将捕获抗体组装在修饰C-dots/nafion复合物薄膜的电极表面，利用差分脉冲伏安技术，通过对比抗原-抗体反应前后检测电流变化的大小来检测目标物质。实验结果表明，在最佳实验条件下，该免疫传感器具有较好的特异性，测得MT-3的浓度线性范围为5pg mL-1~20ng mL-1，其检测限为2pg mL-1，明显低于目前已有的检测MT-3方法的检测限，检测骨肉瘤血清样本的结果与酶联免疫吸附试验（ELISA）的检测结果比较没有显著性差异，结果令人满意。
第三部分：微孔板式化学发光免疫分析法测定人血清中肿瘤坏死因子α(TNF-α)
本章节建立了一种基于微孔板的化学发光免疫分析方法，用于检测人血清中的肿瘤坏死因子α。该方法具有灵敏度高、特异性强、快速、稳定性好、重复性好等特点。对免疫反应的试验条件及各项物理化学参数，如反应温度、温育时间、包被液、稀释度、发光底物的用量等进行了测定和优化。在最佳条件下，该方法的线性范围是10-2000pg/ml，检测限为5.46pg/ml；批内和批间变异系数均小于10%；回收率在90-110%之间，通过本法对实际血清样本进行检测，并将结果与商品化试剂盒的测定结果进行相关分析，其相关系数r=0.9857，证实本法可用于骨肉瘤血清中肿瘤坏死因子α的临床测定。
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 K_3[Fe(CN)_6] assignal for facile and sensitive determination of tumor necrosis factor-alpha
Using K_3[Fe(CN)_6] as the label-free signal material, buiding the electrochemicalimmunosensor which was modified with nafion/K_3[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 K_3[Fe(CN)_6] as redox active signal and C-dots/nafion film for antibodyimmobilization
Building 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 K_3[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 serum
A 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.
骨肉瘤耐药性与其耐药基因表达的标志性耐药蛋白密切相关，通过检测骨肉瘤标志性耐药蛋白，对骨肉瘤病理学分级、化疗过程中的药物监测、疗效评估、快速筛选适合患者的抗肿瘤药物有着极其重要的作用，并可为临床上实现个体化化疗和预后判断提供科学的实验依据。
目前用于骨肉瘤标志性耐药蛋白的临床监测方法主要包括酶联免疫吸附法（ELISA）、免疫组化染色法、基因芯片、RT-PCR等技术，但这些方法均存在一定的局限性，如敏感性不高、操作繁琐、周期长、费用高等问题，迫切需要开发研制一种快速、简便、灵敏、准确、经济的骨肉瘤耐药相关蛋白检测技术，电化学免疫传感器技术及化学发光免疫分析技术为解决这一难题提供了新的出路。
本文设计并研制了两种电化学免疫传感器和一种化学发光免疫分析技术，成功实现了骨肉瘤相关耐药蛋白（肿瘤坏死因子-α和金属硫蛋白-3）检测，为临床提供了新型的检测手段，有助于实现临床上骨肉瘤化疗过程中的实时药物耐药监测，以便及时调整化疗方案，实现骨肉瘤的个体化化疗，大大改善患者的预后。全文共分为三部分，分述如下：
第一部分：基于铁氰化钾作为指示信号的免标记电化学免疫传感器用于肿瘤坏死因子α(TNF-α)的超灵敏检测
本章节主要以K_3[Fe(CN)_6]作为免标记电化学信号物质，构建了nafion/K_3[Fe(CN)_6]-壳聚糖-戊二醛（K-CS-GA）复合物修饰的电化学免疫传感器，根据抗原-抗体反应原理，用于肿瘤坏死因子（TNF-α）的精密检测。利用扫描电子显微镜（SEM）、循环伏安技术（CV）和荧光倒置显微镜等对修饰电极的制备过程进行表征，以及利用紫外可见分光光度法（UV-VIS）和红外分光光度法（FTIR）对制备的修饰复合物K-CS-GA溶液进行表征。实验结果表明，在最佳实验条件下，该方法所制备的nafion-K-C-GCE免疫传感器具有较强的生物大分子识别能力，具有较高的灵敏度和较宽的检测范围，用于检测TNF-α的线性范围为0.02~34ng/ml，其检测限为10pg/ml，用于骨肉瘤血清样本测定结果令人满意，并且该免疫传感器合成方法简单易行，所用修饰复合物K-CS-GA溶液廉价易得并且具有长期稳定性。因此，该电化学免疫传感器具有广阔的临床应用前景。
第二部分：建立基于K_3[Fe(CN)_6]氧化还原信号和C-dots/nafion薄膜固定抗体的电化学免疫传感器用于金属硫蛋白(MT-3)的检测
本章节通过滴涂方式，构建了C-dots/nafion和K-CS-GA复合物修饰的电化学免疫传感器，以K_3[Fe(CN)_6]作为电化学氧化还原信号来源，通过应用C-dots对差分脉冲伏安曲线氧化峰电流信号进行放大，并且以免疫反应为基础，用于金属硫蛋白（MT-3）的精密测定。该方法先将电化学信号活性复合物K-CS-GA和C-dots/nafion复合物依次修饰在处理好的电极上，再将捕获抗体组装在修饰C-dots/nafion复合物薄膜的电极表面，利用差分脉冲伏安技术，通过对比抗原-抗体反应前后检测电流变化的大小来检测目标物质。实验结果表明，在最佳实验条件下，该免疫传感器具有较好的特异性，测得MT-3的浓度线性范围为5pg mL-1~20ng mL-1，其检测限为2pg mL-1，明显低于目前已有的检测MT-3方法的检测限，检测骨肉瘤血清样本的结果与酶联免疫吸附试验（ELISA）的检测结果比较没有显著性差异，结果令人满意。
第三部分：微孔板式化学发光免疫分析法测定人血清中肿瘤坏死因子α(TNF-α)
本章节建立了一种基于微孔板的化学发光免疫分析方法，用于检测人血清中的肿瘤坏死因子α。该方法具有灵敏度高、特异性强、快速、稳定性好、重复性好等特点。对免疫反应的试验条件及各项物理化学参数，如反应温度、温育时间、包被液、稀释度、发光底物的用量等进行了测定和优化。在最佳条件下，该方法的线性范围是10-2000pg/ml，检测限为5.46pg/ml；批内和批间变异系数均小于10%；回收率在90-110%之间，通过本法对实际血清样本进行检测，并将结果与商品化试剂盒的测定结果进行相关分析，其相关系数r=0.9857，证实本法可用于骨肉瘤血清中肿瘤坏死因子α的临床测定。
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 K_3[Fe(CN)_6] assignal for facile and sensitive determination of tumor necrosis factor-alpha
Using K_3[Fe(CN)_6] as the label-free signal material, buiding the electrochemicalimmunosensor which was modified with nafion/K_3[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 K_3[Fe(CN)_6] as redox active signal and C-dots/nafion film for antibodyimmobilization
Building 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 K_3[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 serum
A 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.
引文
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