摘要
本论文以金纳米粒子在食品安全中藻毒素的分析检测和生物检测领域的应用为主线,研究中将具有特异的物理、化学性质的金纳米材料作为探针,与其他传统以及先进的检测技术相结合,建立了针对痕量藻毒素的新颖、快速、超灵敏的分析检测新方法。
首先,通过化学和物理方法将藻毒素抗体和藻毒素-OVA抗原定向的修饰在金纳米棒的端面或侧面,制备的抗体通过竞争免疫反应,形成端面对端面,侧面靠侧面的金纳米棒组装体,通过紫外可见吸收光谱和透射电镜来表征组装体的形貌和性质,通过紫外或动态散射仪的信号和藻毒素浓度之间的关系,建立了检测藻毒素的标准曲线。结果发现端面对端面组装模式的检测结果优于侧面靠侧面的组装模式。这种可控免疫组装方法同样适用于食品中其他相关毒素的检测。
其次,金纳米棒通过修饰抗体和抗原制备高灵敏探针,运用金纳米棒-抗体,金纳米棒-抗原探针的生物识别反应,金纳米棒组装成端面对端面的链状结构。修饰在金纳米棒两端的信号分子的表面增强拉曼信号由于金纳米棒的组装而被增强。目标分子藻毒素的浓度的增度会影响金纳米棒的组装进而影响信号分子的拉曼散射强度。通过透射电镜、紫外吸收光谱和拉曼光谱来表征组装体的变化。最后根据拉曼信号和藻毒素的浓度关系建立标准曲线,实现对藻毒素的超灵敏检测,检测限为0.01ng/mL,高于传统ELISA检测的5倍。
第三,利用G-四链体DNA酶建立免疫检测方法来对藻毒素进行检测。通过G-四链体-血晶素高效的类过氧化物酶活性和金纳米粒子的增强作用,此方法用于检测目标物具有简单、高灵敏和高选择性的特点。包被抗原MC-LR-OVA包被至酶标板孔里,通过和MC-LR抗体和目标分子竞争形成抗原抗体免疫复合物。紧接着,免疫复合物与G-四链体标记的二抗反应,通过比色法测定目标物的含量。该方法对于藻毒素的检测范围与检测限分别是0.1-10ng/mL和0.05ng/mL。该方法可以作为传统ELISA方法的备选方法。
最后,制备了新型的用于输送反义核酸来诱导癌细胞凋亡的碳纳米管和金纳米粒子的复合载体。反义核酸和金纳米粒子共价偶联,然后通过静电作用吸附到碳纳米管侧面上。相比于单独的反义核酸,这个载体系统能够更加有效的转运反义核酸进入细胞,进而发挥促进癌细胞凋亡的效果。金纳米粒子在载体系统中的作用是一方面可以增强转染效果,另外可以提高细胞内的拉曼增强信号,达到追踪癌细胞的效果。
Due to the unique physical and chemical properties of gold nanomaterial, the goldnanomaterials are widely applied for various analytical and biological fields. This dissertationdiscuess with the issues of developing series novel, rapid and sensitive toxin detection methoscombine the gold nanomaterials with the traditional and advanced detection methos.
Firstly, MC-LR detection was successfully achieved with controllable assembly of goldnanorods (GNRs), which are modified either on the sides or ends, using covalent or electrostaticroutes of protein attachment. The side-by-side and end-to-end assembly of the gold NR wererealized using complementary Ab and antigen. Both sensitivity and detection ranges using UV-visand DLS are markedly better for the end-to-end motif. The controllable immunoassembly methodsfollowing the described approach can be applied to a large variety of environmental toxins bysimple modification of the preparation procedure.
Secondly, gold nanorods (GNRs) probes were prepared by modification of GNRs withantibody (Ab) and antigen (Ag, MC-LR-OVA) respective. Using the GNR-Ab and GNR-Ag as theprobes, GNRs were assembled into nanorod chains (end-to-end) through the bio-recognition ofantibody and antigen. The SERS signal of the probe molecule modified to the end of the NRscould be enhanced due to the hot spot between the NRs. The SERS signal could be tuned bychanging the degree of the assembly structure of the NRs. With increased MC-LR concentration inthe solution, the toxin molecules competed with the MC-LR-OVA antigen immobilized on theNRs. We characterized the changes in the degree of nanorod chains by TEM UV-vis and SERS.The limit of detection of MC-LR was0.01ng/mL, and the time necessary for the analysis usingour method (about35min) was decreased by approximately5-fold as compared with traditionalELISA.
Thirdly, we demonstrate the application of versatile G-quadruplex-hemin DNAzymes in animmunoassay for detecting a toxin. Taking advantage of the high peroxidase activity ofG-quadruplex hemin complexes and the enhancement effect of gold nanoparticles (AuNPs), themethod showed simple, high sensitive and selectivity detection of target toxin residues in watersamples. The coated antigen, microcystin-LR (MC-LR)-ovalbumin (OVA), when coated on a plate,competed for MC-LR antibody with added target analyte to form antibody-antigen immunecomplexes. Subsequently, the immune complex reacted with G-quadruplex-labeled secondaryantibodies for colorimetric detection of MC-LR. This assay specifically determined MC-LR in thelinear range of0.1-10ng/mL, with a limit of detection (LOD) for MC-LR of0.05ng/mL. Theresults indicated that the novel immunoassay is an alternative to traditional plate-basedimmunoassays.
Lastly, a novel transfection vector was designed to deliver the antisense oligodeoxynucleotideinto the cancer cells (HL-60). Antisense oligodeoxynucleotides (ASODNs) were conjugated to thegold nanoparticles (GNPs) through a chemical reaction. The conjugates were then bound to thesingle-walled carbon nanotubes (SWNTs). This vector system can effectively for inducedapoptosis of the cancer cells. The gold nanoparticles helped to improve both transfection andRaman signal. This vector system can effectively for induced apoptosis of the cancer cells, andthus has the potential to be used as a cancer therapy.
引文
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