Development of an Interaction Assay between Single-Stranded Nucleic Acids Trapped with Silica Particles and Fluorescent Compounds

Journal of Functional Biomaterials

T. Isoda¹ 

[1] Department of Life and Environment Engineering, Faculty of Environmental Engineering, University of Kitakyushu, 1-1, Hibikino, Wakamatsu, Kitakyushu 808-0135, Japan

关键词: nucleic acid;    polynucleotide;    assay;    fluorescence;    cellstain-AO;   

DOI  :  10.3390/jfb3030601

来源: mdpi

【 摘 要 】

Biopolymers are easily denatured by heating, a change in pH or chemical substances when they are immobilized on a substrate. To prevent denaturation of biopolymers, we developed a method to trap a polynucleotide on a substrate by hydrogen bonding using silica particles with surfaces modified by aminoalkyl chains ([A-AM silane]/SiO₂). [A-AM silane]/SiO₂ was synthesized by silane coupling reaction of N-2-(aminoethyl)-3-aminopropyltrimethoxysilane (A-AM silane) with SiO₂ particles with a diameter of 5 μm at 100 °C for 20 min. The surface chemical structure of [A-AM silane]/SiO₂ was characterized by Fourier transform infrared spectroscopy and molecular orbital calculations. The surface of the silica particles was modified with A-AM silane and primary amine groups were formed. [A-AM silane]/SiO₂ was trapped with single-stranded nucleic acids [(Poly-X; X = A (adenine), G (guanine) and C (cytosine)] in PBS solution at 37 °C for 1 h. The single-stranded nucleic acids were trapped on the surface of the [A-AM silane]/SiO₂ by hydrogen bonding to form conjugated materials. The resulting complexes were further conjugated by derivatives of acridine orange (AO) as fluorescent labels under the same conditions to form [AO:Poly-X:A-AM silane]/SiO₂ complexes. Changes in the fluorescence intensity of these complexes originating from interactions between the single-stranded nucleic acid and aromatic compounds were also evaluated. The change in intensity displayed the order [AO: Poly-G: A-AM silane]/SiO₂ > [AO:Poly-A:A-AM silane]/SiO₂ >> [AO:Poly-C:A-AM silane]/SiO₂. This suggests that the single-stranded nucleic acids conjugated with aminoalkyl chains on the surfaces of SiO₂ particles and the change in fluorescence intensity reflected the molecular interaction between AO and the nucleic-acid base in a polynucleotide.

【 授权许可】

CC BY   
© 2012 by the authors; licensee MDPI, Basel, Switzerland.