title: Polymerase chain reaction coupling with magnetic nanoparticles-based biotin-avidin system for amplification of chemiluminescent detection signals of nucleic acid.
abstract: 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 probes were immobilized on the surface of silanized MNPs by Schiff reaction between 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 (SA-AP). 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 AP. The concentration of probes which were immobilized on the surface of MNPs was studied, how to reduce the adsorption of SA-AP on the surface of MNPs was also researched. It was shown that 12.5 pmol of probes were immobilized on 1 mg of MNPs. Aldehyde-MNPs modified with probes could adsorb SA-AP, 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.
authors: State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
authors: Zhiyang
authors: Z
authors: Li
authors: Lei
authors: L
authors: He
authors: Nongyue
authors: N
authors: He
authors: Yan
authors: Y
authors: Deng
authors: Zhiyang
authors: Z
authors: Shi
authors: Hua
authors: H
authors: Wang
authors: Song
authors: S
authors: Li
authors: Hongna
authors: H
authors: Liu
authors: Zhifei
authors: Z
authors: Wang
authors: Daxin
authors: D
authors: Wang
mesh_terms: D000818:Animals; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002417:Cattle; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D036103:Nanotechnology; D009696:Nucleic Acids / Q000032:analysis* / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012710:Serum Albumin, Bovine
chemical_list: D058185:Magnetite Nanoparticles; D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D001360:Avidin; D012710:Serum Albumin, Bovine; D001710:Biotin; D015500:Chloramphenicol O-Acetyltransferase