纳米粒子免疫传感器及其在癌细胞早期检测和免疫分析中的应用
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摘要
医学免疫分析分为体液免疫分析和细胞免疫分析两大部分。体液免疫分析是测定体液中游离在寄主细胞外的抗原及其产生的有毒物质,如病毒,细菌等；细胞免疫分析则是测定细胞膜表面的特异性蛋白或侵入到寄主细胞内的病毒、胞内寄生菌或外来的组织团块、癌变的细胞等。免疫分析要求高亲合力、高特异性和超高灵敏度检测。为了实现这一目标,本论文研究用细胞融合技术制备高亲合力和特异性的单克隆抗体,用高量子产率的荧光纳米粒子作标记物,和用荧光显微成像；激光诱导荧光阵列分析和化学发光阵列分析进行检测。用以上研究制备的纳米粒子免疫传感器已经成功地用于结肠癌细胞、7721肝癌细胞及HHCC肝癌细胞的细胞免疫分析,实现对这些癌细胞的灵敏检测,配合流式细胞技术进行癌细胞早期诊断。采用细胞融合技术制备了一对能同特异蛋白质上两种不同位点进行识别的高亲合力的单克隆抗体。实现了对金黄色葡萄球菌肠毒素C1及胱抑素C高灵敏和高特异性免疫分析。具体研究内容和结果如下：
(1)通过三步水解法制备了氨基化的荧光化学发光双功能介孔二氧化硅纳米粒子。纳米通道内的疏水基团能有效的防止染料的泄漏。表面的氨基使该纳米粒子更易于生物功能化。由于纳米二氧化硅中独立的纳米通道能削弱荧光染料分子间的内滤效应能,从而有效地减弱荧光分子的猝灭。得到了90nm的二氧化硅粒子的量子产率约为61%。相比核壳荧光纳米颗粒,化学发光试剂可以自由进入纳米粒子与荧光染料反应产生化学发光。结果表明,FCMSN是荧光标记和化学发光标记。在生物学中的应用,过碘酸钠氧化法被证明优于戊二醛法。表面氨基的量影响FCMSN在生物应用中的特异性。
(2)建立了一种采用抗CD155和抗CD112单克隆抗体标记的荧光共振能量转移介孔二氧化硅纳米颗粒(FCMSN)检测肝癌细胞的方法。罗丹明6G与荧光素被同时掺杂到介孔二氧化硅纳米粒子中。得到的纳米颗粒的直径为90nm。量子产率为69%。由于荧光素的发射光谱与罗月明6G的激发峰高度重叠,且在纳米粒子中两种染料足够接近,所以两种染料之间会发生荧光共振能量转移。纳米孔道和二氧化硅网络骨架能有效削弱染料的内滤效应,也使激发峰更宽,增加染料的有效载荷量。更宽的斯托克位移减弱了激发光的干扰,同时提高检测的灵敏度。单克隆抗体的共价固定化采用不使用交联剂的过碘酸钠氧化法。这些优点使得检测SMMC-7721肝癌细胞HHCC细胞的纳米传感器具有较高的灵敏度和特异性。
(3)通过将抗人上皮细胞粘附分子(EpCAM)单克隆抗体抗体共价修饰到核壳型二氧化硅纳米粒子上制备了一种敏感的和有选择性的检测结肠癌细胞的纳米传感器。透射电子显微镜(TEM)图像表明,纳米粒子和生物功能化的纳米粒子具有良好的分散性。过碘酸钠氧化的方法很好地保持了活性的抗体。荧光显微成像和流式细胞仪(FCM)的实验表明,纳米传感器能明显提高信号强度和区分三种靶细胞(Colo205, SW480和NCM460)。核膜双染色表明纳米传感器在细胞膜表面结合。最后,一个简单体系的示例证明抗EpCAM抗体纳米传感器能有效地区分Colo205细胞,并为后续研究打下了良好的基础。
(4)建立了敏感的金黄色葡萄球菌肠毒素C1(SEC1)免疫分析。氨基化的染料掺杂的介孔二氧化硅纳米颗粒(FCMSN)作为标记物。由于染料和疏水基团之间的疏水相互作用,染料被有效固定在FCMSN的矩阵中。在FCMSN表面的胺基团提高了纳米颗粒的标记性质。使用双抗体夹心法,单克隆抗体(G8)和单克隆抗体(C4)分别作为捕获抗体和检测抗体。TCPO体系用来进行化学发光检测。激光诱导荧光则用来进行荧光检测。结果表明,FCMSN(?)能够起到有效放大信号的作用。化学发光检测线性范围为0.025-2ng/mL,检出限0.019ng/ml (3σ)。工作曲线的回归方程为I=3027.5[SEC1](ng/mL)+1804.6(R2=0.9958)。11次平行测量1ng/ml SEC的相对标准偏差(R.S.D.)为4.6%。激光诱导荧光的线性范围0.05-2ng/mL,检出限0.042ng/mL,回归方程I=302.5[SEC1](ng/mL)+672.6(R2=0.9908)。测定lng/mL SECl (N=11)的相对标准偏差(R.S.D.)为5.3%。此外,全自动化学发光分析仪能有效提高提高检测效率。为SEC1提供了敏感、快速、简便的检测方法。
(5)胱抑素C是人体内一个重要的半胱氨酸蛋白酶抑制剂。在肾损伤检测中,它是新的检测肾小球滤过率的标志物。在这章中,利用功能化的FCMSN作为标记物,采用染料溶出化学发光夹心免疫分析法对胱抑素C进行了检测。该法具有检出限低,简单快速的特点。氨基官能化的染料封装FCMSN采用三步水解法制备。纳米基质具内的疏水性环境用于固定染料。纳米粒子表面的氨基基团可以用于生物标记。此外,过碘酸钠标记法很好地保持了抗体活性。分析表明,该法线性范围0.0025-0.5ng/mL (R2=0.9936),检出限0.0020ng/mL。11次平行测定0.25ng/mL CysC的相对标准偏差(R.S.D.)为4.2%。
本研究主要特色和创新点为：
1.采用三步水解法,在极稀的表面活性剂浓度下制备了形貌较好的罗丹明6G掺杂的介孔二氧化硅纳米粒子。MTMS水解后的甲基使介孔和基质内部产生疏水环境从而实现对染料的固定。APTES水解产生的氨基使介孔二氧化硅纳米粒子更易于标记且增加了纳米粒子在水中的分散性。制得的介孔二氧化硅纳米粒子具有很高的量子产率。同时,由于罗丹明6G既能产生荧光又是过氧化草酸酯-过氧化氢-咪唑化学发光体系的能量转移体且介孔结构对溶液传质影响比核壳结构小的多,使得制备的介孔二氧化硅纳米粒子可以同时作为荧光标记物和化学发光标记物,实现单标记物的双功能化。还利用上述方法制备了罗丹明6G与荧光素双掺杂的荧光共振能量转移介孔二氧化硅纳米粒子。由于荧光素的发射峰与罗丹明6G的激发峰有较大重叠,且二氧化硅纳米粒子基质能保证两种染料足够接近,所以荧光素和罗丹明6G可以发生荧光共振能量转移。和单染料掺杂相比,该法扩大了荧光介孔二氧化硅纳米粒子的斯托克斯位移,能削弱激发光对检测的影响。荧光量子产率也较单染料有所增加。
2.比较了过碘酸钠氧化法与戊二醛法在抗体固定化方面的优劣。由于过碘酸钠氧化法不使用交联剂,避免了交联剂对抗体活性的影响,提高了纳米传感器与目标抗原的结合能力。在染料掺杂介孔二氧化硅纳米粒子的共价修饰方面,直接过碘酸钠氧化法还避免了交联剂将染料从介孔二氧化硅纳米粒子中溶出的缺点。
3.将抗人上皮细胞粘附分子单克隆抗体共价固定在掺杂了Rubpy的核壳型二氧化硅纳米粒子上,并对结肠癌细胞进行了特异性检测。荧光成像的结果能直观的说明细胞表面抗原的分布与数量,但对极低表达的细胞效果有限。流式细胞分析能量化比较细胞表面抗原数量并提高了辨识低表达细胞的能力。
4.使用荧光介孔二氧化硅纳米粒子对肝癌细胞进行了成像研究。荧光显微成像结果直观定性地显示了CD155与CD112在SMMC-7721肝癌细胞与HHCC肝癌细胞表面的数量差别。此外,发现介孔二氧化硅纳米粒子表面氨基的量对介孔二氧化硅纳米粒子的特异性也有一定的影响。
5.与第四军医大学免疫教研室合作,采用细胞融合技术制备了一对能同金黄色葡萄球菌肠毒素C1特异蛋白质上两种不同位点进行识别的高亲合力的单克隆抗体(G8和C4),用双染料掺杂介孔二氧化硅纳米粒子作为标记物,采用TCPO体系对金黄色葡萄球菌肠毒素C1进行了化学发光检测。由于介孔二氧化硅纳米粒子能负载大量染料分子,该法检出限达到了0.019ng/ml。在测定胱抑素C时,为了减小二氧化硅基质对传质的影响,采用了染料溶出法。即在化学发光检测前先将介孔二氧化硅纳米粒子内的染料溶出,再进行化学发光测定。和直接法相比,该法进一步地降低了检出限。
Medical immune analysis consists of humoral immunity and cellular immunity analysis of two most analysis. Humoral immune analysis is a toxic substance, free in the host extracellular antigen and its determination in body fluids, such as viruses, bacteria; cell immune analysis is the determination of the cell membrane surface specific protein or invading into the host cell, the virus intracellular bacteria or foreign organizations, cancerous cells. Immune analysis requires high affinity, high specificity and high sensitivity detection. In order to achieve this goal, this paper study on the preparation of monoclonal antibodies with high affinity and specificity for cell fusion technique, marking with fluorescent nanoparticles with high quantum yield, and fluorescence microscopy imaging; laser induced fluorescence array analysis and chemiluminescence array analysis in detecting. Nanoparticle immune sensor for the above research preparation has been successfully used for analysis of cell immune colon cancer,7721liver cancer cells and HHCC tumor cells, for sensitive detection of these cancer cells, with the early diagnosis of cancer cell flow cytometry. The specific protein with two different sites identification of high affinity monoclonal antibody was prepared by the cell fusion technique. The high sensitivity of Staphylococcus aureus enterotoxin C1and cystatin C and high specificity immune analysis were devoloped. Specifically described as follows:
(1) A new kind of ultrabright fluorescent and chemiluminescent difunctional mesoporous silica nanoparticle (FCMSN) is reported. A luminescent dye, Rhodamine6G or tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy), is doped inside nanochannels of a silica matrix. The hydrophobic groups in the silica matrix avoid the leakage of dye from open channels. The amines groups on the surface of the FCMSN improve the modification performance of the nanoparticle. Because the nanochannels are isolated by a network skeleton of silica, fluorescence quenching based on the inner filter effect of the fluorescent dyes immobilized in nanochannels is weakened effectively. The Quantum Yield of obtained90nm silica particles was about61%. Compared with the fluorescent core-shell nanoparticle, the chemiluminescence reagents can freely enter the nanoparticles to react with fluorescent dyes to create chemiluminescence. The results show that the FCMSN are both fluorescent labels and chemiluminescent labels. In biological applications, the NaIO4oxidation method was proven to be superior to the glutaraldehyde method. The amount of amino could affect the specificity of the FCMSN. The fluorescence microscopy imaging demonstrated that the FCMSN is viable for biological applications.
(2) A novel method for sensitive detection of liver cancer cells using anti-CD155and anti-CD112monoclonal antibodies conjugated ultrabright fluorescent mesoporous silica nanoparticle (FCMSN) encapsulating Rhodamine6G and fluorescein has been developed. The diameter of obtained nanoparticle was90nm. The quantum yield of FCMSN was69%. Because the emission of fluorescein has a high degree of overlap with excitation of Rhodamine6G, and the two kinds of dyes are close enough in the nanoparticle, the fluorescence resonance energy transfer occurs between two kinds of dyes. It not only maintains the original feature which the nanochannels and network skeleton of silica weaken the inner filter of dye, but also makes the excitation peak wider and increases useful load amount of dye. Because the wider stokes shift weaken the interference of excitation, the detecting sensitivity is enhanced at the same time. The NaIO4oxidation method, which didn't use cross-linker, was used covalent immobilization of monoclonal antibodies on the FCMSN. This method can maintain the activity of the monoclonal antibodies more easily than the glutaraldehyde method. These advantages ensure that nanosensor has high sensitivity and specificity for detecting liver cancer SMMC-7721cell and HHCC cell.
(3) A sensitive and selective sensor for detecting colon cancer cells based on nanoparticle covalent modified anti-human epithelial cell adhesion molecule (EpCAM) antibody is developed. The transmission electron microscope (TEM) images showed that the nanoparticle and functionalized nanoparticle had good decentrality for application. The NaIO4oxidation method, which was used oxidizing antibody for immobilization of conjugating antibody on the silica-coated fluorescent nanoparticles, maintained the activities of antibodies very well. The fluorescence microscopy imaging and flow cytometer (FCM) experiments demonstrated that the nanosensor could increase the signal intensity obviously and distinguish three kinds of target cells (colo205, sw480and NCM460) well. The membrane and nuclear staining showed the distribution and abundance of EpCAM in cells' membrane. It also provides a possibility to quantify special membrane proteins on different regions of cells'surface. At the end, the result of detecting a simple sample proved that colo205cells were selected by anti-EpCAM antibody nanosensors in this environment, and made a good foundation for subsequent research.
(4) Sensitive chemiluminescence immunoassay and laser induced fluorescence immunoassay for staphylococcal enterotoxin C1(SEC1) based on the functionalized dye-encapsulated mesoporous silica nanoparticle (FCMSN) as label has been developed. Because of hydrophobic interaction between the dyes and the hydrophobic groups, the dyes are fixed in the MSN's matrix effectively. The amines groups on the surface of the FCMSN improve the labeling properties of the nanoparticles. In the sandwich immunoassay, the capturing monoclonal antibody (G8) and the detecting monoclonal antibody (C4) were used. In chemiluminescence detection steps, TCPO system was adopted. The results showed that the functionalized MSN played the role of signal amplification effectively. The range of detecting SEC1was0.025-2ng/mL with a detection limit of0.019ng/ml (3σ). The regression equation of the working curve is I=3O27.5[SEC1](ng/mL)+1804.6(R2=0.9958). The relative standard deviation (R.S.D.) for11parallel measurements of1ng/ml SEC1was4.6%. The performance of laser induced fluorescence immunoassay was0.05-2ng/mL,0.042ng/mL,I=302.5[SEC1](ng/mL)+672.6(R2=0.9908). R.S.D. was5.3%after detecting lng/mL SEC1(N=11). Furthermore, fully automated chemiluminescence analyzer improves the detection efficiency. The results demonstrated that these methods offer potential advantages of sensitivity, simplicity and rapidity for detecting SEC1.
(5) Cystatin C is a significant cysteine protease inhibitor in human bodies, and is proposed as a fascinating novel marker of glomerular filtration rate for kidney injury detection. In this article, we report an ultra-sensitive, simple, rapid chemiluminescence immunoassays method for cystatin C detection using functionalized MSN. After a three step hydrolysis, the amino-functionalized MSN encapsulating dye has hydrophobic environment for fixing dye and amino groups for biologic modification. The NaIO4immobilization method maintains the activity of antibody very well. The sandwich immunoassay using two monoclonal antibodies was chosen for its selectivity. The analysis showed a linear response within the range of0.0025-0.5ng/mL (R2=0.9936). The relative standard deviation (R.S.D.) for11parallel measurements of0.25ng/mL CysC was4.2%. The automated chemiluminescence analyzer can detect96well continuously. The results proved that this method is ultra-sensitive, simple, and rapid for detecting cystatin C
(1)通过三步水解法制备了氨基化的荧光化学发光双功能介孔二氧化硅纳米粒子。纳米通道内的疏水基团能有效的防止染料的泄漏。表面的氨基使该纳米粒子更易于生物功能化。由于纳米二氧化硅中独立的纳米通道能削弱荧光染料分子间的内滤效应能,从而有效地减弱荧光分子的猝灭。得到了90nm的二氧化硅粒子的量子产率约为61%。相比核壳荧光纳米颗粒,化学发光试剂可以自由进入纳米粒子与荧光染料反应产生化学发光。结果表明,FCMSN是荧光标记和化学发光标记。在生物学中的应用,过碘酸钠氧化法被证明优于戊二醛法。表面氨基的量影响FCMSN在生物应用中的特异性。
(2)建立了一种采用抗CD155和抗CD112单克隆抗体标记的荧光共振能量转移介孔二氧化硅纳米颗粒(FCMSN)检测肝癌细胞的方法。罗丹明6G与荧光素被同时掺杂到介孔二氧化硅纳米粒子中。得到的纳米颗粒的直径为90nm。量子产率为69%。由于荧光素的发射光谱与罗月明6G的激发峰高度重叠,且在纳米粒子中两种染料足够接近,所以两种染料之间会发生荧光共振能量转移。纳米孔道和二氧化硅网络骨架能有效削弱染料的内滤效应,也使激发峰更宽,增加染料的有效载荷量。更宽的斯托克位移减弱了激发光的干扰,同时提高检测的灵敏度。单克隆抗体的共价固定化采用不使用交联剂的过碘酸钠氧化法。这些优点使得检测SMMC-7721肝癌细胞HHCC细胞的纳米传感器具有较高的灵敏度和特异性。
(3)通过将抗人上皮细胞粘附分子(EpCAM)单克隆抗体抗体共价修饰到核壳型二氧化硅纳米粒子上制备了一种敏感的和有选择性的检测结肠癌细胞的纳米传感器。透射电子显微镜(TEM)图像表明,纳米粒子和生物功能化的纳米粒子具有良好的分散性。过碘酸钠氧化的方法很好地保持了活性的抗体。荧光显微成像和流式细胞仪(FCM)的实验表明,纳米传感器能明显提高信号强度和区分三种靶细胞(Colo205, SW480和NCM460)。核膜双染色表明纳米传感器在细胞膜表面结合。最后,一个简单体系的示例证明抗EpCAM抗体纳米传感器能有效地区分Colo205细胞,并为后续研究打下了良好的基础。
(4)建立了敏感的金黄色葡萄球菌肠毒素C1(SEC1)免疫分析。氨基化的染料掺杂的介孔二氧化硅纳米颗粒(FCMSN)作为标记物。由于染料和疏水基团之间的疏水相互作用,染料被有效固定在FCMSN的矩阵中。在FCMSN表面的胺基团提高了纳米颗粒的标记性质。使用双抗体夹心法,单克隆抗体(G8)和单克隆抗体(C4)分别作为捕获抗体和检测抗体。TCPO体系用来进行化学发光检测。激光诱导荧光则用来进行荧光检测。结果表明,FCMSN(?)能够起到有效放大信号的作用。化学发光检测线性范围为0.025-2ng/mL,检出限0.019ng/ml (3σ)。工作曲线的回归方程为I=3027.5[SEC1](ng/mL)+1804.6(R2=0.9958)。11次平行测量1ng/ml SEC的相对标准偏差(R.S.D.)为4.6%。激光诱导荧光的线性范围0.05-2ng/mL,检出限0.042ng/mL,回归方程I=302.5[SEC1](ng/mL)+672.6(R2=0.9908)。测定lng/mL SECl (N=11)的相对标准偏差(R.S.D.)为5.3%。此外,全自动化学发光分析仪能有效提高提高检测效率。为SEC1提供了敏感、快速、简便的检测方法。
(5)胱抑素C是人体内一个重要的半胱氨酸蛋白酶抑制剂。在肾损伤检测中,它是新的检测肾小球滤过率的标志物。在这章中,利用功能化的FCMSN作为标记物,采用染料溶出化学发光夹心免疫分析法对胱抑素C进行了检测。该法具有检出限低,简单快速的特点。氨基官能化的染料封装FCMSN采用三步水解法制备。纳米基质具内的疏水性环境用于固定染料。纳米粒子表面的氨基基团可以用于生物标记。此外,过碘酸钠标记法很好地保持了抗体活性。分析表明,该法线性范围0.0025-0.5ng/mL (R2=0.9936),检出限0.0020ng/mL。11次平行测定0.25ng/mL CysC的相对标准偏差(R.S.D.)为4.2%。
本研究主要特色和创新点为：
1.采用三步水解法,在极稀的表面活性剂浓度下制备了形貌较好的罗丹明6G掺杂的介孔二氧化硅纳米粒子。MTMS水解后的甲基使介孔和基质内部产生疏水环境从而实现对染料的固定。APTES水解产生的氨基使介孔二氧化硅纳米粒子更易于标记且增加了纳米粒子在水中的分散性。制得的介孔二氧化硅纳米粒子具有很高的量子产率。同时,由于罗丹明6G既能产生荧光又是过氧化草酸酯-过氧化氢-咪唑化学发光体系的能量转移体且介孔结构对溶液传质影响比核壳结构小的多,使得制备的介孔二氧化硅纳米粒子可以同时作为荧光标记物和化学发光标记物,实现单标记物的双功能化。还利用上述方法制备了罗丹明6G与荧光素双掺杂的荧光共振能量转移介孔二氧化硅纳米粒子。由于荧光素的发射峰与罗丹明6G的激发峰有较大重叠,且二氧化硅纳米粒子基质能保证两种染料足够接近,所以荧光素和罗丹明6G可以发生荧光共振能量转移。和单染料掺杂相比,该法扩大了荧光介孔二氧化硅纳米粒子的斯托克斯位移,能削弱激发光对检测的影响。荧光量子产率也较单染料有所增加。
2.比较了过碘酸钠氧化法与戊二醛法在抗体固定化方面的优劣。由于过碘酸钠氧化法不使用交联剂,避免了交联剂对抗体活性的影响,提高了纳米传感器与目标抗原的结合能力。在染料掺杂介孔二氧化硅纳米粒子的共价修饰方面,直接过碘酸钠氧化法还避免了交联剂将染料从介孔二氧化硅纳米粒子中溶出的缺点。
3.将抗人上皮细胞粘附分子单克隆抗体共价固定在掺杂了Rubpy的核壳型二氧化硅纳米粒子上,并对结肠癌细胞进行了特异性检测。荧光成像的结果能直观的说明细胞表面抗原的分布与数量,但对极低表达的细胞效果有限。流式细胞分析能量化比较细胞表面抗原数量并提高了辨识低表达细胞的能力。
4.使用荧光介孔二氧化硅纳米粒子对肝癌细胞进行了成像研究。荧光显微成像结果直观定性地显示了CD155与CD112在SMMC-7721肝癌细胞与HHCC肝癌细胞表面的数量差别。此外,发现介孔二氧化硅纳米粒子表面氨基的量对介孔二氧化硅纳米粒子的特异性也有一定的影响。
5.与第四军医大学免疫教研室合作,采用细胞融合技术制备了一对能同金黄色葡萄球菌肠毒素C1特异蛋白质上两种不同位点进行识别的高亲合力的单克隆抗体(G8和C4),用双染料掺杂介孔二氧化硅纳米粒子作为标记物,采用TCPO体系对金黄色葡萄球菌肠毒素C1进行了化学发光检测。由于介孔二氧化硅纳米粒子能负载大量染料分子,该法检出限达到了0.019ng/ml。在测定胱抑素C时,为了减小二氧化硅基质对传质的影响,采用了染料溶出法。即在化学发光检测前先将介孔二氧化硅纳米粒子内的染料溶出,再进行化学发光测定。和直接法相比,该法进一步地降低了检出限。
Medical immune analysis consists of humoral immunity and cellular immunity analysis of two most analysis. Humoral immune analysis is a toxic substance, free in the host extracellular antigen and its determination in body fluids, such as viruses, bacteria; cell immune analysis is the determination of the cell membrane surface specific protein or invading into the host cell, the virus intracellular bacteria or foreign organizations, cancerous cells. Immune analysis requires high affinity, high specificity and high sensitivity detection. In order to achieve this goal, this paper study on the preparation of monoclonal antibodies with high affinity and specificity for cell fusion technique, marking with fluorescent nanoparticles with high quantum yield, and fluorescence microscopy imaging; laser induced fluorescence array analysis and chemiluminescence array analysis in detecting. Nanoparticle immune sensor for the above research preparation has been successfully used for analysis of cell immune colon cancer,7721liver cancer cells and HHCC tumor cells, for sensitive detection of these cancer cells, with the early diagnosis of cancer cell flow cytometry. The specific protein with two different sites identification of high affinity monoclonal antibody was prepared by the cell fusion technique. The high sensitivity of Staphylococcus aureus enterotoxin C1and cystatin C and high specificity immune analysis were devoloped. Specifically described as follows:
(1) A new kind of ultrabright fluorescent and chemiluminescent difunctional mesoporous silica nanoparticle (FCMSN) is reported. A luminescent dye, Rhodamine6G or tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy), is doped inside nanochannels of a silica matrix. The hydrophobic groups in the silica matrix avoid the leakage of dye from open channels. The amines groups on the surface of the FCMSN improve the modification performance of the nanoparticle. Because the nanochannels are isolated by a network skeleton of silica, fluorescence quenching based on the inner filter effect of the fluorescent dyes immobilized in nanochannels is weakened effectively. The Quantum Yield of obtained90nm silica particles was about61%. Compared with the fluorescent core-shell nanoparticle, the chemiluminescence reagents can freely enter the nanoparticles to react with fluorescent dyes to create chemiluminescence. The results show that the FCMSN are both fluorescent labels and chemiluminescent labels. In biological applications, the NaIO4oxidation method was proven to be superior to the glutaraldehyde method. The amount of amino could affect the specificity of the FCMSN. The fluorescence microscopy imaging demonstrated that the FCMSN is viable for biological applications.
(2) A novel method for sensitive detection of liver cancer cells using anti-CD155and anti-CD112monoclonal antibodies conjugated ultrabright fluorescent mesoporous silica nanoparticle (FCMSN) encapsulating Rhodamine6G and fluorescein has been developed. The diameter of obtained nanoparticle was90nm. The quantum yield of FCMSN was69%. Because the emission of fluorescein has a high degree of overlap with excitation of Rhodamine6G, and the two kinds of dyes are close enough in the nanoparticle, the fluorescence resonance energy transfer occurs between two kinds of dyes. It not only maintains the original feature which the nanochannels and network skeleton of silica weaken the inner filter of dye, but also makes the excitation peak wider and increases useful load amount of dye. Because the wider stokes shift weaken the interference of excitation, the detecting sensitivity is enhanced at the same time. The NaIO4oxidation method, which didn't use cross-linker, was used covalent immobilization of monoclonal antibodies on the FCMSN. This method can maintain the activity of the monoclonal antibodies more easily than the glutaraldehyde method. These advantages ensure that nanosensor has high sensitivity and specificity for detecting liver cancer SMMC-7721cell and HHCC cell.
(3) A sensitive and selective sensor for detecting colon cancer cells based on nanoparticle covalent modified anti-human epithelial cell adhesion molecule (EpCAM) antibody is developed. The transmission electron microscope (TEM) images showed that the nanoparticle and functionalized nanoparticle had good decentrality for application. The NaIO4oxidation method, which was used oxidizing antibody for immobilization of conjugating antibody on the silica-coated fluorescent nanoparticles, maintained the activities of antibodies very well. The fluorescence microscopy imaging and flow cytometer (FCM) experiments demonstrated that the nanosensor could increase the signal intensity obviously and distinguish three kinds of target cells (colo205, sw480and NCM460) well. The membrane and nuclear staining showed the distribution and abundance of EpCAM in cells' membrane. It also provides a possibility to quantify special membrane proteins on different regions of cells'surface. At the end, the result of detecting a simple sample proved that colo205cells were selected by anti-EpCAM antibody nanosensors in this environment, and made a good foundation for subsequent research.
(4) Sensitive chemiluminescence immunoassay and laser induced fluorescence immunoassay for staphylococcal enterotoxin C1(SEC1) based on the functionalized dye-encapsulated mesoporous silica nanoparticle (FCMSN) as label has been developed. Because of hydrophobic interaction between the dyes and the hydrophobic groups, the dyes are fixed in the MSN's matrix effectively. The amines groups on the surface of the FCMSN improve the labeling properties of the nanoparticles. In the sandwich immunoassay, the capturing monoclonal antibody (G8) and the detecting monoclonal antibody (C4) were used. In chemiluminescence detection steps, TCPO system was adopted. The results showed that the functionalized MSN played the role of signal amplification effectively. The range of detecting SEC1was0.025-2ng/mL with a detection limit of0.019ng/ml (3σ). The regression equation of the working curve is I=3O27.5[SEC1](ng/mL)+1804.6(R2=0.9958). The relative standard deviation (R.S.D.) for11parallel measurements of1ng/ml SEC1was4.6%. The performance of laser induced fluorescence immunoassay was0.05-2ng/mL,0.042ng/mL,I=302.5[SEC1](ng/mL)+672.6(R2=0.9908). R.S.D. was5.3%after detecting lng/mL SEC1(N=11). Furthermore, fully automated chemiluminescence analyzer improves the detection efficiency. The results demonstrated that these methods offer potential advantages of sensitivity, simplicity and rapidity for detecting SEC1.
(5) Cystatin C is a significant cysteine protease inhibitor in human bodies, and is proposed as a fascinating novel marker of glomerular filtration rate for kidney injury detection. In this article, we report an ultra-sensitive, simple, rapid chemiluminescence immunoassays method for cystatin C detection using functionalized MSN. After a three step hydrolysis, the amino-functionalized MSN encapsulating dye has hydrophobic environment for fixing dye and amino groups for biologic modification. The NaIO4immobilization method maintains the activity of antibody very well. The sandwich immunoassay using two monoclonal antibodies was chosen for its selectivity. The analysis showed a linear response within the range of0.0025-0.5ng/mL (R2=0.9936). The relative standard deviation (R.S.D.) for11parallel measurements of0.25ng/mL CysC was4.2%. The automated chemiluminescence analyzer can detect96well continuously. The results proved that this method is ultra-sensitive, simple, and rapid for detecting cystatin C
引文
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