Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Anal Chem. 2012 May 1;84(9):4192-8. doi: 10.1021/ac300778s. Epub 2012 Apr 18.
A molecular beacon (MB) is comprised of a fluorophore and a quencher linked by a DNA hairpin. MBs have been widely used for homogeneous DNA detection. In addition to molecular quenchers, many nanomaterials such as graphene oxide (GO) also possess excellent quenching efficiency. Most reported fluorescent sensors relied on DNA probes physisorbed by GO, which may suffer from nonspecific probe displacement and false positive signal. In this work, we report the preparation and characterization of a MB using graphene oxide (GO) as quencher, where an amino and FAM (6-carboxyfluorescein) dual labeled DNA was covalently attached to GO via an amide linkage. A major challenge was to remove noncovalently attached probes due to strong DNA adsorption by GO. While DNA desorption was favored at low salt, high pH, high temperature, and by using organic solvents, the cDNA was required to achieve complete desorption of noncovalently linked DNA probes. The DNA adsorption energy was measured using isothermal titration calorimetry, revealing the heterogeneous nature of GO. The covalent probe has a detection limit of 2.2 nM using a sample volume of 0.05 mL. With a 2 mL sample, the detection limit can reach 150 pM. The covalent probe is highly resistant to nonspecific probe displacement and will find applications in serum and cellular samples where high probe stability is demanded.
分子信标（MB）由荧光团和通过 DNA 发夹连接的猝灭剂组成。MB 已被广泛用于均相 DNA 检测。除了分子猝灭剂外，许多纳米材料，如氧化石墨烯（GO），也具有优异的猝灭效率。大多数报道的荧光传感器依赖于通过 GO 物理吸附的 DNA 探针，这可能会受到非特异性探针位移和假阳性信号的影响。在这项工作中，我们报告了一种使用氧化石墨烯（GO）作为猝灭剂的 MB 的制备和表征，其中通过酰胺键将氨基和 FAM（6-羧基荧光素）双重标记的 DNA 共价连接到 GO 上。一个主要的挑战是去除由于 GO 对 DNA 的强吸附而非共价连接的探针。虽然 DNA 解吸在低盐、高 pH、高温和有机溶剂存在的情况下是有利的，但需要 cDNA 来实现非共价连接的 DNA 探针的完全解吸。使用等温滴定量热法测量 DNA 吸附能，揭示了 GO 的非均相性质。使用 0.05 mL 样品体积，共价探针的检测限为 2.2 nM。使用 2 mL 样品，检测限可达到 150 pM。共价探针对非特异性探针位移具有高度抗性，将在需要高探针稳定性的血清和细胞样品中得到应用。
