Development of chemiluminescent biosensing of nucleic acids based on oligonucleotide-immobilized gold surfaces

A chemiluminescent biosensing method was developed based on the immobilization of DNA probes on a gold surface by the self-assembled monolayers technique. The complementary sequence was detected by coupling avidin-alkaline phosphatase to the biotinylated oligonucleotide and measuring the chemiluminescent signal obtained from the hydrolysis of the substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane by this enzyme. The method has a wide calibration range of five orders of magnitude and the detection limit is 15 pM (signal-to-ratio = 3). The factors affecting the probe immobilization, target hybridization and sensitivity were investigated. The method was also applied to detect specific DNA fragments relative to the hepatitis B plasmid.