Patent ID: 8962240
Filing Date: 2015-02-24
Classification: C12Q

Abstract:
1. A method for electrochemically identifying a predetermined target nucleotide sequence in a biological sample by real-time detection, comprising: providing the biological sample; providing material for activatable amplification comprising free nucleotides used for replication of the predetermined target nucleotide sequence and for the formation of replicated target nucleotide sequences; bringing an oxido-reducible compound capable of intercalating between the nucleotides forming the replicated target sequences, and which does not oxidize the nucleotide bases of the free nucleotides in the biological sample, into contact with the biological sample and the provided material for activatable amplification, the oxido-reducible compound comprising a metal and at least one ligand, the metal being selected from the group consisting of osmium and ruthenium, and the at least one ligand being selected from the group consisting of dipyrido[3,2-a:2′,3′-c]phenazine, 7,8-(dimethyl)dipyrido[3,2-a:2,3-c]phenazine (DPPZ), 3-(pyridin-2-yl)-5,6-diphenyl-as-triazine (PTDB), 3-(pyrazin-2-yl)-as-triazino[5,6-f]phenanthrene (DPT), and phenanthrenequinone diimine (PHI); activating the material for activatable amplification, in a series of amplification cycles, to successively increase a quantity of double stranded replicated target sequences dependent on a quantity of the predetermined target nucleotide sequence in the biological sample, wherein a portion of the oxido-reducible compound intercalates between the nucleotides forming the replicated target sequences after formation, the intercalated portion of the oxido-reducible compound having inhibited electrochemical activity with respect to a non-intercalated portion; applying an electric field during each cycle of amplification while the portion of the oxido-reducible compound intercalates between the nucleotides forming the replicated target sequences, to selectively measuring an electric current representative of the electrochemical activity of the portion of the oxido-reducible compound that is non-intercalated, distinguished from the intercalated portion of the oxido-reducible compound; and quantitatively determining the presence of the predetermined target nucleotide sequence based on the measurement of the electric current during a time when the portion of the oxido-reducible compound is intercalated with the double stranded replicated target sequences, wherein the replication of the predetermined target nucleotide sequence by activating the material for activatable amplification causes a reduction in the portion of the intercalated oxido-reducible compound which is not intercalated, resulting in a quantitative decrease in the electric current as the fraction of intercalated oxido-reducible compound increases.