化学学报 ›› 2014, Vol. 72 ›› Issue (9): 1012-1016.DOI: 10.6023/A14050348 上一篇    下一篇

研究论文

寡聚(4-乙烯基苯基磷酸)/金纳米粒子的磷酸酶可视化检测研究

范艳斌a,b, 陈道勇a,b   

  1. a 复旦大学高分子科学系 上海 200433;
    b 聚合物分子工程国家重点实验室 上海 200433
  • 收稿日期:2014-05-04 出版日期:2014-09-14 发布日期:2014-08-27
  • 通讯作者: 陈道勇 E-mail:chendy@fudan.edu.cn
  • 基金资助:

    项目受国家自然科学基金(Nos. 21334001,91127030)及科技部973项目基金(No. 2011CB932503)资助

Oligo(4-Vinyl-Phenyl Phosphate)/Gold Nanoconjugate for Visual Detection of Phosphatases

Fan Yanbina,b, Chen Daoyonga,b   

  1. a Department of Macromolecular Science, Fudan University, Shanghai 200433;
    b State Key Laboratory of Molecular Engineering of Polymer, Shanghai 200433
  • Received:2014-05-04 Online:2014-09-14 Published:2014-08-27
  • Supported by:

    Project supported by the National Natural Science Foundation of China (Nos. 21334001 and 91127030) and the Ministry of Science and Technology of China (No. 2011CB932503).

基于金属纳米粒子的磷酸酶可视化检测技术具有重要的生物医用前景. 然而, 文献中报道的方法大多采用多肽或核酸等生物分子作为磷酸酶感应基团. 由于这些生物分子在水溶液中不稳定, 且可被磷酸酶以外的生物酶类降解, 导致这些方法在实际应用中存在局限性. 首次合成了一种化学结构稳定的水溶性寡聚(4-乙烯基苯基磷酸)[oligo(4-vinyl-phenyl phosphate), OVPP], 并制备得到由OVPP稳定的磷酸酶响应型金纳米粒子Au@OVPP, 最后以酸性磷酸酶为例, 考察了Au@OVPP在磷酸酶可视化检测方面的应用. 由于OVPP的亲水性, Au@OVPP可在水溶液中稳定分散; 当体系中存在磷酸酶时, 由于OVPP中的亲水基团(磷酸根)被磷酸酶水解释放, 使得Au@OVPP的稳定分子OVPP由亲水变为疏水, 进而导致金纳米粒子的聚集及溶液宏观颜色的改变. 通过(肉眼)观察Au@OVPP溶液的变色及程度, 建立了一种灵敏、专一和低成本的磷酸酶检测方法. 该方法对于酸性磷酸酶的(肉眼)检测下限约为10 U/mL, 且对多种生物分子具有较好的抗干扰能力.

关键词: 金纳米粒子, 磷酸衍生物, 磷酸酶, 可视化, 检测

Colorimetric assay taking advantage of the unique surface plasmon resonance properties of metallic nanoparticles has attracted significant attention in bioassays due to its simplicity, low cost, high sensitivity and compatibility. However, most reported colorimetric methods used adenosine triphosphate (ATP), peptides or nucleotides (DNA and RNA) as the phosphatase-sensing species, which are quite unstable or hydrolysable by other enzymes in normal conditions. In this paper, a chemically stable and hydrophilic oligo(4-vinyl-phenyl phosphate) (OVPP) was synthesized. Specifically, the oligomer precursor oligo(dibenzyl-4-vinylphenyl phosphate) was synthesized via RAFT polymerization of a synthetic monomer dibenzyl-4-vinylphenyl phosphate in dioxane, using S-ethyl-S'-(α,α'-dimethyl-α"-acetic acid) trithiocarbonate as the chain transfer agent. Deprotection by trimethylsilyl bromide in deuterochloroform containing 2,4,6-collidine at 0 ℃ and then hydrolysis in water at pH 9.0 gives the desired OVPP. Then, an OVPP-stabilized and phosphatase-responsive gold nanoparticle was prepared via simple ligand exchange in water and finally tested for visual detection of a model phosphatase acid phosphatase (ACP). Due to the hydrophilicity of OVPP, Au@OVPP is soluble and stable in water. In the presence of ACP, however, the hydrophilic phosphate groups in OVPP were hydrolyzed and released, transforming the hydrophilic OVPP of Au@OVPP into hydrophobic type. This change from hydrophilicity to hydrophobicity triggered the aggregation and color changes of the gold nanoparticles and their sols, respectively. Via this method, a sensitive visual detection of ACP was achieved, with a detection limit of ca. 10 U/mL after 24 h incubation at pH 5.0 and 37 ℃. Besides, a high selectivity for the phosphatase detection was confirmed as controlled with other bio-species including esterase, proteinase, serum proteins, DNA and so on. By using pyrene as a polarity probe, a significant decrease of the ratio I373/I384 was found in Au@OVPP sols after incubation with ACP, indicating the hydrolysis of OVPP and formation of hydrophobic domains in the ACP activated solutions. Collectively, Au@OVPP represents a chemically stable, low cost, highly sensitive and specific phosphatase-visualization agent for phosphatase detection.

Key words: gold nanoparticle, phosphate derivate, phosphatase, visualization, detection