基于新型纳米发光和磁性材料的甲胎蛋白临床检测试剂盒的研发
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摘要
肝癌是一种高危险性的恶性肿瘤,被称为当今世界的三大肿瘤杀手之一。因此,开发一种快速而廉价的检测方法,对提前预测肝癌并及时采取治疗措施显得尤为必要。目前,预测是否患有肝癌的常用手段是检测血清中的甲胎蛋白(Alpha-fetoprotein,称α-FP或AFP)量。而检测AFP浓度的常用方法为酶联免疫沉淀分析、免疫印迹、和免疫荧光分析。然而,这些方法不但耗时耗力,而且需要经过训练的专业人员进行操作,还需要先进的仪器设备。
在过去的几十年中,半导体量子点(通常被称为量子点,简写为QDs)CdTe已经引起了广泛的关注,CdTe具有的诸多优点使它比传统的染料具有更明显的优势,更适合于生物医学领域的应用。同时,磁性纳米粒子由于其本身特性在生物医学领域的应用也得到推广。但是,结合这这两种纳米粒子的本身优势的具体应用还很少报道。
首先,本文将阐述CdTe量子点的制备,并对其制备过程进行优化,然后把CdTe量子点和模型蛋白——牛血清白蛋白(BSA)——结合。CdTe的制备采用巯基水相法,即利用L—型半胱胺氨酸(L-Cysteine,简称L-Cys)作为稳定剂,在水相中进行合成。同时对CdTe量子点的制备过程中具有至关重要作用的因素做出系统研究,这些因素包括反应时间、反应前驱体中环境pH值,反应物浓度以及反应物摩尔比等,并通过对这一系列反应条件的改变,获得制备CdTe量子点的最佳条件。同时,将制备出的CdTe量子点与模型蛋白BSA结合,观察结合前后荧光强度的变化,结果显示,CdTe量子点可以作为一种很好的蛋白质探针。
其次,本文将阐述制备出顺磁性良好的Fe_3O_4-dextran(右旋糖酐)磁性纳米粒子,并对这种磁性纳米粒子的分散性做出表征,观察它的分散性。使用蒽酮比色法检测Fe_3O_4-dextran磁性纳米粒子所携带的右旋糖酐的量。结果发现,1mg的磁性纳米粒子表面包含的右旋糖酐的量约为390μg,这说明Fe_3O_4-dextran纳米粒子表面携带足够的右旋糖酐,使用碘酸钾(KIO_4)将右旋糖酐的羟基氧化为醛基,这样醛基就能够比较容易和蛋白质的氨基相互作用生成共价键,最终实现Fe_3O_4纳米粒子和蛋白质分子的共价结合。这为下一步将抗体结合在Fe_3O_4-dextran表面打下基础。
最后,本文将阐述如何制备标准工作曲线、推算标准工作方程以及临床血液样品的分析,其步骤为:(1)将制备好的Fe_3O_4-dextran纳米粒子氧化,然后和鼠抗人AFP抗体(记为第一抗体)进行结合,得到“Fe_3O_4—第一抗体”复合物。使用这种复合物与一系列已知标准浓度的抗原结合,通过磁分离对抗原进行分离纯化,得到“磁性纳米粒子—第一抗体—抗原”复合物。(2)制备水溶性荧光纳米粒子,并将其与另外一种鼠抗人AFP抗体(区别于第一抗体,与第一抗体识别抗原的位点不一样,记为第二抗体)结合,得到“CdTe-第二抗体”复合物。(3)将“CdTe-第二抗体”复合物和“Fe_3O_4—第一抗体—抗原”复合物作用,得到三明治复合物。结合磁分离,洗去未结合的“CdTe-第二抗体”复合物,然后将产物定容至1ml并测荧光,得到荧光强度和抗原浓度的对应关系,绘制出“荧光强度—抗原浓度”工作曲线,并根据该工作曲线,算出标准工作方程:Y=k(X+4.5)/V。(4)使用同样的方法处理临10例床血液样品。实验结果发现,当血液样本的血清中AFP含量较高的时候,本实验所得到的检测结果和使用时间分辨荧光检测所得到的结果较为相似,当血液样本的血清中AFP含量较低的时候,本实验所得到的检测结果和使用时间分辨荧光检测所得到的结果偏差较大。出现这种结果可能的原因是受到本体干扰。
由于条件限制,本课题不能随意采集到人体血样,期望在后续的研究中,能够在采血过程中注意血液的前处理,减少处理不当带来的本体干扰。同时,纳米粒子的制备过程有待进一步优化,以制备出半坡宽更窄的CdTe量子点和单分散性更好的Fe_3O_4-dextran磁性纳米粒子等。
本课题所述的方法同样适用于其它“抗原—抗体”体系的应用,只要更换相应的抗体,即可对相应的抗原进行定量分析。
Hepatocellular carcinoma(HCC)is a highly malignant cancer,being the third cause of tumor-related death in the world.Thus,there must be excellent candidates for the rapid and inexpensive diagnosis of diseases.Recently,the usual way to predict HCC is detecting the concentration of AFP in serum and the most common approach in analyzing AFP is an enzyme-linked immunosorbent assay immunoprecipitation,immunoblotting techniques,and immunofluorescence.However,most of the usual methods are time-consuming,labor-intensive, and hazardous to health or require highly qualified personnel and sophisticated instrumentation.
In the past decades,semiconductor nanocrystals(often known as quantum dots,QDs)have been attracted wide interests due to their various beneficial factors,which makes them take advantage over the convenient dyes,and more suitable for the usage in biology and medicine. Meanwhile,magnetic nanoparticles had been thoroughly studied due to their widespread biomedical applications.Up to now,however,there have been few researches in which QDs and magnetic nanoparticles were both used in practical application.
In this paper,firstly,CdTe QDs were synthesized by aqueous phase method,and the preparation course was optimized.Then the CdTe QDs were conjugated with the bovine serum albumin(BSA).The preparation of CdTe was occurred in the aqueous phase by using L-Cysteine as stabilizer.The influence of reflux time,pH of precursor,and the mole ratio of reagent was investigated respectively to get the optimum reaction conditions.The CdTe QDs were conjugated with the BSA through electrostatic attrationand.The PL intensity of the complex was tested so as to ensure that the CdTe QDs were appropriate to be used as a protein probe.
Secondly,Fe_3O_4-dextran magnetic nanoparticles with good paramagnetism were prepared and their monodispersity was checked by using transmission electron microscope.The amount of anthrone in the surface magnetic nanoparticles was determined by the anthrone colorimetry method.The result showed that about 390μg of dextran was absorbed in the surface of 1 mg of Fe_3O_4-dextran,suggesting that there was enough dextran in the surface of each magnetic nanoparticle.After the hydroxyl of dextran was oxidized into aldehyde group by KIO_4,the Fe_3O_4-dextran nanoparticles could easily conjugate with protein.
Finally,the calibration curve and the working equation were obtained.Based on the equation, the human serums were tested.The processes were:(1)the Fe_3O_4-dextran nanoparticles were oxidized and conjugated with mouse anti-AFP antibody(the primary antibody)firstly.And then the complexes of primary antibody and magnetic nanoparticle were used to capture the antigen, AFP,so as to separate them.(2)The water soluble fluorescent nanoparticles were synthesized and conjugated with mouse anti-AFP antibody(the second antibody).(3)The sandwich immunoreaction occurred,and the superfluous QDs-anti-AFP antibody was washed away.The relationship between PL intensity of the complex and the concentration of AFP was determined, and the calibration equation—Y=k(X+4.5)/V—was obtained.(4)The human serum samples of 50 people were tasted.The result showed thatthe outcomes determined by this method were similar to those determined by Time-resolved Fluorescence when the concentration of AFP was high.When the concentration of AFP was low,the difference was slightly obvious.The reason may lie in that there were some molecules with the similar structure to AFP.
Because of inevitable conditional restriction,the human serum could never be collected easily.Sothe pre-treatment of the serum should never be shook so as to reduce the interference causing by the protein molecules with the similar structure to AFP's if the proceeding research were carried out.At the same time,the preparation of nanoparticles waited to be further optimized. Through these ways,the CdTe QDs that had narrower half high,and Fe_3O_4-dextran magnetic nanoparticles better paramagnetism.
More importantly,this new technique could be used in other antibody-antigen system if the antibody was changed.
在过去的几十年中,半导体量子点(通常被称为量子点,简写为QDs)CdTe已经引起了广泛的关注,CdTe具有的诸多优点使它比传统的染料具有更明显的优势,更适合于生物医学领域的应用。同时,磁性纳米粒子由于其本身特性在生物医学领域的应用也得到推广。但是,结合这这两种纳米粒子的本身优势的具体应用还很少报道。
首先,本文将阐述CdTe量子点的制备,并对其制备过程进行优化,然后把CdTe量子点和模型蛋白——牛血清白蛋白(BSA)——结合。CdTe的制备采用巯基水相法,即利用L—型半胱胺氨酸(L-Cysteine,简称L-Cys)作为稳定剂,在水相中进行合成。同时对CdTe量子点的制备过程中具有至关重要作用的因素做出系统研究,这些因素包括反应时间、反应前驱体中环境pH值,反应物浓度以及反应物摩尔比等,并通过对这一系列反应条件的改变,获得制备CdTe量子点的最佳条件。同时,将制备出的CdTe量子点与模型蛋白BSA结合,观察结合前后荧光强度的变化,结果显示,CdTe量子点可以作为一种很好的蛋白质探针。
其次,本文将阐述制备出顺磁性良好的Fe_3O_4-dextran(右旋糖酐)磁性纳米粒子,并对这种磁性纳米粒子的分散性做出表征,观察它的分散性。使用蒽酮比色法检测Fe_3O_4-dextran磁性纳米粒子所携带的右旋糖酐的量。结果发现,1mg的磁性纳米粒子表面包含的右旋糖酐的量约为390μg,这说明Fe_3O_4-dextran纳米粒子表面携带足够的右旋糖酐,使用碘酸钾(KIO_4)将右旋糖酐的羟基氧化为醛基,这样醛基就能够比较容易和蛋白质的氨基相互作用生成共价键,最终实现Fe_3O_4纳米粒子和蛋白质分子的共价结合。这为下一步将抗体结合在Fe_3O_4-dextran表面打下基础。
最后,本文将阐述如何制备标准工作曲线、推算标准工作方程以及临床血液样品的分析,其步骤为:(1)将制备好的Fe_3O_4-dextran纳米粒子氧化,然后和鼠抗人AFP抗体(记为第一抗体)进行结合,得到“Fe_3O_4—第一抗体”复合物。使用这种复合物与一系列已知标准浓度的抗原结合,通过磁分离对抗原进行分离纯化,得到“磁性纳米粒子—第一抗体—抗原”复合物。(2)制备水溶性荧光纳米粒子,并将其与另外一种鼠抗人AFP抗体(区别于第一抗体,与第一抗体识别抗原的位点不一样,记为第二抗体)结合,得到“CdTe-第二抗体”复合物。(3)将“CdTe-第二抗体”复合物和“Fe_3O_4—第一抗体—抗原”复合物作用,得到三明治复合物。结合磁分离,洗去未结合的“CdTe-第二抗体”复合物,然后将产物定容至1ml并测荧光,得到荧光强度和抗原浓度的对应关系,绘制出“荧光强度—抗原浓度”工作曲线,并根据该工作曲线,算出标准工作方程:Y=k(X+4.5)/V。(4)使用同样的方法处理临10例床血液样品。实验结果发现,当血液样本的血清中AFP含量较高的时候,本实验所得到的检测结果和使用时间分辨荧光检测所得到的结果较为相似,当血液样本的血清中AFP含量较低的时候,本实验所得到的检测结果和使用时间分辨荧光检测所得到的结果偏差较大。出现这种结果可能的原因是受到本体干扰。
由于条件限制,本课题不能随意采集到人体血样,期望在后续的研究中,能够在采血过程中注意血液的前处理,减少处理不当带来的本体干扰。同时,纳米粒子的制备过程有待进一步优化,以制备出半坡宽更窄的CdTe量子点和单分散性更好的Fe_3O_4-dextran磁性纳米粒子等。
本课题所述的方法同样适用于其它“抗原—抗体”体系的应用,只要更换相应的抗体,即可对相应的抗原进行定量分析。
Hepatocellular carcinoma(HCC)is a highly malignant cancer,being the third cause of tumor-related death in the world.Thus,there must be excellent candidates for the rapid and inexpensive diagnosis of diseases.Recently,the usual way to predict HCC is detecting the concentration of AFP in serum and the most common approach in analyzing AFP is an enzyme-linked immunosorbent assay immunoprecipitation,immunoblotting techniques,and immunofluorescence.However,most of the usual methods are time-consuming,labor-intensive, and hazardous to health or require highly qualified personnel and sophisticated instrumentation.
In the past decades,semiconductor nanocrystals(often known as quantum dots,QDs)have been attracted wide interests due to their various beneficial factors,which makes them take advantage over the convenient dyes,and more suitable for the usage in biology and medicine. Meanwhile,magnetic nanoparticles had been thoroughly studied due to their widespread biomedical applications.Up to now,however,there have been few researches in which QDs and magnetic nanoparticles were both used in practical application.
In this paper,firstly,CdTe QDs were synthesized by aqueous phase method,and the preparation course was optimized.Then the CdTe QDs were conjugated with the bovine serum albumin(BSA).The preparation of CdTe was occurred in the aqueous phase by using L-Cysteine as stabilizer.The influence of reflux time,pH of precursor,and the mole ratio of reagent was investigated respectively to get the optimum reaction conditions.The CdTe QDs were conjugated with the BSA through electrostatic attrationand.The PL intensity of the complex was tested so as to ensure that the CdTe QDs were appropriate to be used as a protein probe.
Secondly,Fe_3O_4-dextran magnetic nanoparticles with good paramagnetism were prepared and their monodispersity was checked by using transmission electron microscope.The amount of anthrone in the surface magnetic nanoparticles was determined by the anthrone colorimetry method.The result showed that about 390μg of dextran was absorbed in the surface of 1 mg of Fe_3O_4-dextran,suggesting that there was enough dextran in the surface of each magnetic nanoparticle.After the hydroxyl of dextran was oxidized into aldehyde group by KIO_4,the Fe_3O_4-dextran nanoparticles could easily conjugate with protein.
Finally,the calibration curve and the working equation were obtained.Based on the equation, the human serums were tested.The processes were:(1)the Fe_3O_4-dextran nanoparticles were oxidized and conjugated with mouse anti-AFP antibody(the primary antibody)firstly.And then the complexes of primary antibody and magnetic nanoparticle were used to capture the antigen, AFP,so as to separate them.(2)The water soluble fluorescent nanoparticles were synthesized and conjugated with mouse anti-AFP antibody(the second antibody).(3)The sandwich immunoreaction occurred,and the superfluous QDs-anti-AFP antibody was washed away.The relationship between PL intensity of the complex and the concentration of AFP was determined, and the calibration equation—Y=k(X+4.5)/V—was obtained.(4)The human serum samples of 50 people were tasted.The result showed thatthe outcomes determined by this method were similar to those determined by Time-resolved Fluorescence when the concentration of AFP was high.When the concentration of AFP was low,the difference was slightly obvious.The reason may lie in that there were some molecules with the similar structure to AFP.
Because of inevitable conditional restriction,the human serum could never be collected easily.Sothe pre-treatment of the serum should never be shook so as to reduce the interference causing by the protein molecules with the similar structure to AFP's if the proceeding research were carried out.At the same time,the preparation of nanoparticles waited to be further optimized. Through these ways,the CdTe QDs that had narrower half high,and Fe_3O_4-dextran magnetic nanoparticles better paramagnetism.
More importantly,this new technique could be used in other antibody-antigen system if the antibody was changed.
引文
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