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Polymerase Chain Reaction Coupling with Magnetic Nanoparticles-Based Biotin–Avidin System for Amplification of Chemiluminescent Detection Signals of Nucleic Acid

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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.

Keywords: BIOELECTRONICS; BIOTIN-AVIDIN; CHEMILUMINESCENT DETECTION; MAGNETIC NANOPARTICLES; NUCLEIC ACID HYBRIDIZATION

Document Type: Research Article

DOI: https://doi.org/10.1166/jnn.2011.3061

Publication date: 2011-02-01

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  • Journal for Nanoscience and Nanotechnology (JNN) is an international and multidisciplinary peer-reviewed journal with a wide-ranging coverage, consolidating research activities in all areas of nanoscience and nanotechnology into a single and unique reference source. JNN is the first cross-disciplinary journal to publish original full research articles, rapid communications of important new scientific and technological findings, timely state-of-the-art reviews with author's photo and short biography, and current research news encompassing the fundamental and applied research in all disciplines of science, engineering and medicine.
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