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.