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