title: Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for detection of Exotoxin A gene sequence.
abstract: Bio-barcode DNA based on gold nanoparticle (bDNA-GNPs) as a new generation of biosensor based detection tools, holds promise for biological science studies. They are of enormous importance in the emergence of rapid and sensitive procedures for detecting toxins of microorganisms. Exotoxin A (ETA) is the most toxic virulence factor of Pseudomonas aeruginosa. ETA has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. In the present study, we developed a fluorescence bio-barcode technology to trace P. aeruginosa ETA. The GNPs were coated with the first target-specific DNA probe 1 (1pDNA) and bio-barcode DNA, which acted as a signal reporter. The magnetic nanoparticles (MNPs) were coated with the second target-specific DNA probe 2 (2pDNA) that was able to recognize the other end of the target DNA. After binding the nanoparticles with the target DNA, the following sandwich structure was formed: MNP 2pDNA/tDNA/1pDNA-GNP-bDNA. After isolating the sandwiches by a magnetic field, the DNAs of the probes which have been hybridized to their complementary DNA, GNPs and MNPs, via the hydrogen, electrostatic and covalently bonds, were released from the sandwiches after dissolving in dithiothreitol solution (DTT 0.8M). This bio-barcode DNA with known DNA sequence was then detected by fluorescence spectrophotometry. The findings showed that the new method has the advantages of fast, high sensitivity (the detection limit was 1.2ng/ml), good selectivity, and wide linear range of 5-200ng/ml. The regression analysis also showed that there was a good linear relationship (∆F=0.57 [target DNA]+21.31, R2=0.9984) between the fluorescent intensity and the target DNA concentration in the samples.
authors.affiliation: Department of Biology, Science and Research Branch, Islamic Azad University (IAU), Tehran, Iran.
authors.forename: Bahram
authors.initials: B
authors.lastname: Amini
authors.affiliation: Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. Electronic address: mehkamali@yahoo.co.uk.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Kamali
authors.affiliation: Biology Research Center, Zanjan Branch, Islamic Azad University, Zanjan, Iran. Electronic address: saloutim@yahoo.com.
authors.forename: Mojtaba
authors.initials: M
authors.lastname: Salouti
authors.affiliation: Department of Biology, Science and Research Branch, Islamic Azad University (IAU), Tehran, Iran.
authors.forename: Parichehreh
authors.initials: P
authors.lastname: Yaghmaei
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D015374:Biosensing Techniques / Q000379:methods; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D005098:Exotoxins / Q000235:genetics*; D006046:Gold / Q000737:chemistry*; D006801:Humans; D057230:Limit of Detection; D058185:Magnetite Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D011552:Pseudomonas Infections / Q000175:diagnosis / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000379:methods; D037521:Virulence Factors / Q000235:genetics*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D015342:DNA Probes; D004269:DNA, Bacterial; D005098:Exotoxins; D006046:Gold; D058185:Magnetite Nanoparticles; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
keywords: Bio-barcode DNA; Exotoxin A; Gold nanoparticles; Pseudomonas aeruginosa