CHEMILUMINESCENT DETECTION OF NUCLEIC ACIDS BY MEANS OF MAGNETIC NANOPARTICLES AND PCR

A novel method was established through the detection of chemiluminescent signals of nucleic acid hybridization based on magnetic nanoparticles (MNPs) and PCR. 5' amino-modified specific probe was immobilized on the surface of silanized MNPs by Schiff reaction of amino and aldehyde group. The probes were used to capture the synthetic biotin-dUTP-labeled DNA fragments which were obtained by polymerase chain reaction (PCR). Then these complexes were bonded with streptavidin-modified alkaline phosphatase (ALP-SA). Finally the chemiluminescent signals were detected by adding 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy) phenyl - 1, 2 - dioxetane (AMPPD) which was the substrate reagent of ALP. The concentration of probes which were immobilized on the surface of MNPs was studied, how to reduce the absorption of avidin - ALP on the surface of MNPs was also researched. It was showed that 12.5 pmol probes were immobilized on 1 mg MNPs. Aldehyde MNPs modified with probe could adsorb avidin-modified ALP, affecting the sensitivity of chemiluminescene consequently. Reduction of aldehyde group by sodium borohydride and blocking the bare position of MNPs with bovine serum albumin (BSA) could decrease the background of chemiluminescence, and this method has good specificity in detection of chloramphenicol acetyltransferase (CAT) gene.