氧化石墨烯脂质体的制备及其在化学发光生物分析中的应用

It was reported that graphene oxide (GO) showed peroxidase-like activity, based on which GO was expected to replace natural peroxidase (HRP) and serve as new marker in chemiluminescent bioanalysis. However, it was found that GO was unstable during the labeling process, and the labeled GO exhibited much lower catalytic activity than that of unlabeled GO. The above disadvantages restrict the application of GO as novel marker in chemiluminescent bioanalysis. Liposome, which has large specific surface area and a large internal cavity, is formed spontaneously when phospholipids are dispersed into water. In this project, GO loading immunoliposomes are prepared by encapsulating nano-sized GO into internal cave of liposomes and conjugating biological recognition molecules on the surface of liposomes. The prepared GO loading immunoliposomes will be used to develop chemiluminescent bioanalysis methods for the determination of anthrax related sequence-specific DNA, serum tumor marker (CEA) and small cell lung cancer circulating tumor cells. The proposed method has several advantages: 1) GO is encapasulated in the internal cave of liposomes avoiding the problems of GO being conjugated with biological recognition molecules. 2) Because of their extensive internal volume, liposomes can carry a high number of nano-sized GO. Therefore, higher detection sensitivity can be obtained by using GO loading liposomes as labels. 3) The proposed methods can be easily extended to the determination of other sequence-specific DNA, proteins, tumor cells, or even small molecules, thus, exhibit potential in a wide range of applications.

氧化石墨烯（GO）具有拟过氧化物酶活性，有望取代天然过氧化物酶（HRP）成为新型的化学发光生物分析标记物。但GO在标记过程中易聚集，且标记后的催化活性显著降低，制约了GO在化学发光生物分析中的应用。脂质体是一种在水相中由磷脂双分子层定向排列而形成的囊泡体，不仅具有大的内水腔，且具有较大的比表面积。课题拟将纳米尺寸的GO碎片，包裹入脂质体的内水腔，制备GO脂质体，将生物识别分子偶联于脂质体表面，并将制备的GO免疫脂质体应用于炭疽杆菌特定序列DNA、肿瘤血清标志物癌胚抗原（CEA）及小细胞肺癌循环肿瘤细胞的化学发光测定。制备的GO免疫脂质体具有以下优势：1）可解决GO偶联后化学发光催化活性大大降低的问题；2）一个脂质体可包入多个GO，可显著提高目标分析物的检测灵敏度；3）制备的GO免疫脂质体极易拓宽至其他特定序列DNA、蛋白质生物大分子，肿瘤细胞，甚至是小分子的检测，具有广泛的应用价值。

寻找能够替代HRP 的模拟酶和催化体系是一个具有重要意义的研究领域，已逐渐成为各国学者的研究热点。氧化石墨烯具有良好的化学发光催化活性，但标记后催化活性将大幅降低。课题按照研究计划制备了纳米氧化石墨烯，选用薄膜分散法制备了氧化石墨烯脂质体，所制备的氧化石墨烯脂质体的粒径约为400 nm，粒径均一，包封率为40%，4 ℃条件下的稳定性较好。以制备的氧化石墨烯脂质体为标记物，课题建立了化学发光免疫分析法测定兔IgG。但是，我们发现以氧化石墨烯脂质体为标记物，测定兔IgG的线性范围为50-500 ng，检测灵敏度只能达到10 ng，并未达到实验所预期的检测灵敏度提高的效果。分析原因：氧化石墨烯脂质体形成过程中，纳米氧化石墨烯并未如预期进入脂质体的内水腔，而可能与磷脂形成复合物，这可能改变氧化石墨烯的表面性质，而导致其学发光催化活性降低。虽然仍可以产生化学发光信号，但是测定目标物的检测灵敏度低。课题进一步考察了磷脂与氧化石墨烯的相互作用及磷脂对氧化石墨烯化学发光催化活性的影响，发现当氧化石墨烯与脂质体以合适比例制备得到复合物时，采用曲拉通100将脂质体破膜后，可恢复氧化石墨烯的化学发光催化活性，因此发展了以功能化脂质体与氧化石墨烯形成复合物，并以所形成的复合物为标记物，建立了癌胚抗原的化学发光免疫分析方法。除此之外，课题利用氧化石墨烯对多肽和单链DNA的吸附作用，构建了无标记的化学发光分析体系检测胰蛋白酶的活性和血清中ATP的含量。课题除了聚焦于氧化石墨烯的化学发光催化活性，还建立了5种新型的化学发光催化体系，发现金纳米簇、银纳米簇和阳离子化金纳米簇均有良好的化学发光催化活性，并将其应用于了过氧化氢、血清中葡萄糖、尿酸等的检测。课题发表相关SCI论文8篇，其中影响因子3.0以上4篇，他引50次，获得专利一项，培养硕士研究生7名。