Alginic acid-based macromolecular chemiluminescent probe for universal protein assay on a solid-phase membrane

Abstract

A novel chemiluminescent (CL) technique for the rapid determination of proteins on a membrane is described. The method utilizes an interaction between luminol-labeled alginic acid macromolecule and proteins. The synthesis of the macromolecular probe consists of the oxidation of alginic acid with NaIO₄, the introduction of luminol through imine formation as a CL tag, and the reduction of the conjugate with NaBH₄ to obtain the stable probe. The analytical protocol consists of adsorbing proteins on a poly(vinylidene difluoride) (PVDF) membrane, incubating the membrane for 30 min with the probe solution in the presence of boric acid and a surfactant, two short washing steps in order to remove an excess of the probe, and detection of CL intensity with hemin, tetra-n-propylammonium hydroxide and H₂O₂. This proposed CL assay for proteins can be finished within 1 h, and indicates the detection limit of 15–250 ng of proteins on the membrane. The CL signals in the calibration curves for some proteins such as albumin show proportional intensities against the amounts of the proteins less than ca. 125 ng, though there is a logarithmic relationship between the CL signals and the protein amounts larger than ca. 125 ng. However, some other proteins indicate the proportional CL intensities against the increasing amounts in wider range up to 500 ng of the proteins. The synthesised alginic acid-based probe indicates specific selectivity towards proteins, and should be used as a CL probe for the universal detection of various proteins on a solid-phase membrane even in the presence of DNA and RNA.