title: Construction, electrochemically biosensing and discrimination of recombinant plasmid (pEThIL-2) on the basis of interleukine-2 DNA insert.
abstract: Construction, electrochemically biosensing and discrimination of recombinant pEThIL-2 plasmid, with 5839bp size, on the basis of interleukine-2 (IL-2) DNA insert are described. Plasmid pEThIL-2 was constructed by PCR amplification of IL-2 encoding DNA and subcloning into pET21a(+) vector using BamHI and SacI sites. The recombinant pEThIL-2 plasmid was detected with a label-free DNA hybridization biosensor using a non-inosine substituted probe. The proposed sensor was made up by immobilization of a 20-mer antisense single strand oligonucleotide (chIL-2) related to the human interleukine-2 gene on the pencil graphite electrode (PGE) as a probe and then the sensing of recombinant pEThIL-2 plasmid was conducted by anodic differential pulse voltammetry (ADPV) based on guanine oxidation signal. Selectivity of the detection was assessed with pET21a(+) non-complementary plasmid, with 5443bp size, lacking IL-2 encoding DNA. Different factors such as electrode activation conditions and washing strategy were tested in order to eliminate the nonspecific adsorption of pET21a(+). We have found that the PGE activation for 300s produces a condition in which desorption of nonspecifically adsorbed plasmids from the electrode surface can be achieved by 300s washing of the electrode in 20mM Tris-HCl buffer solution (pH 7.0) containing 20mM NaCl. Diagnostic performance of the biosensor is described and the detection limit is found to be 10.31pg/microL.
authors.affiliation: Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Mohammad Saeid
authors.initials: MS
authors.lastname: Hejazi
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Pournaghi-Azar
authors.forename: Esmaeel
authors.initials: E
authors.lastname: Alipour
authors.forename: Farrokh
authors.initials: F
authors.lastname: Karimi
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D004251:DNA Transposable Elements / Q000235:genetics; D004563:Electrochemistry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007376:Interleukin-2 / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D010957:Plasmids / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D004251:DNA Transposable Elements; D007376:Interleukin-2