Label-Free Aptamer Nanogold Resonance Scattering Method for Trace Alkaline Phosphatase Coupling the Adenosine Triphosphate Reaction

Abstract

In pH 8.5 Tris-HCl buffer solutions, alkaline phosphatase (ALP) catalyzed the hydrolysis of adenosine triphosphate (ATP) substrate to form adenosine diphosphate (ADP) that can be terminated by addition of H₃PO₄. The unhydrolyzed ATP molecules react specifically with the DNA1 of double strand DNA (dsDNA) to produce a stable G-quartet and a single stranded DNA2 (DNA 2) that can protect nanogold (NG) to form stable NG-DNA2 conjugates. In the presence of salt, the unprotected NGs were combined to form large aggregates that exhibited a resonance scattering (RS) peak at 590 nm. Under the chosen conditions, when ALP increased, the ATP decreased, the formed DNA2 decreased, that is, the NG-DNA2 decreased, and the NG aggregations increased that caused the RS peak to be enhanced linearly at 590 nm. The enhanced RS intensity ΔI was linear to ALP content in the range of 0.2–7.83 µM, with a detection limit of 0.1 µM ALP. This enzyme-catalyzed aptamer-nanogold RS method was applied to analysis of real samples, and the results were in agreement with those of a fluorescence method.

Keywords:

• Adenosine triphosphate
• Alkaline phosphatase
• Gold nanoparticle
• Label-free aptamer
• Resonance scattering spectral method

Notes

Note: SP, salicylic phosphate; PNPP, nitrophenylphosphate-alkaline phosphatase; MU, 4-methyl umbelliferone; QP, 8-quinoline phosphate; CNP, 2-carboxyl-1-naphthyl phosphate; EC, enzyme catalytic; AAP, ascorbic acid 2-phosphate; ANG, aptamer NG.

Note: *The human serum was provided by the Fifth Hospital of Guilin.