Employment of bromophenol red and bovine serum albumin as luminol signal co-enhancer in chemiluminescent detection of sequence-specific DNA

Abstract

Bromophenol red, known as chemical indicator, was found to act as a novel potent signal enhancer of the peroxidase-catalyzed luminol–H2O2 chemiluminescent (CL) reaction. It was found interestingly that bovine serum albumin (BSA) played a role in the enhanced chemiluminescent reaction (ECR). The addition of 2.5mgmL−1 BSA into bromophenol red-enhance CL system showed 36 times stronger CL signal than that without addition of BSA. Mechanism study showed that the luminophors in the ECR were still 3-aminophthalate ion in an excited state (3-APA*). In addition, singlet oxygen (1O2) and hydroxyl radical (∙OH) played a role in the ECR. The possible mechanism was discussed in the present study. The effect of pH, reaction time, and concentration of bromophenol red, BSA, luminol, and H2O2 on CL intensity of the peroxidase-catalyzed CL reaction was studied. The detection limit value (LOD) of HRP and streptavidin-modified HRP in the proposed ECR with bromophenol red and BSA was 0.20ngmL−1 and 0.05ngmL−1, respectively. This novel luminol–H2O2–HRP–bromophenol red-BSA CL system was applied to the CL detection of sequence-specific DNA based on a magnetic separation process. As low as 0.4fmol of target DNA could be sensitively detected using the proposed CL system without any amplification process. The obtained results demonstrate very promising perspectives for using bromophenol red and BSA to improve the sensitivity of CL detection of sequence-specific DNA. In addition, this novel ECR system can also be generalized for CL immunoassay, CL western blotting, and so on.