Studies On Electrochemical DNA Biosensors Based On G-quadruplex DNAzyme

Detection of trace sequence-specific DNA has shown significant applications in the field of clinic diagnosis, gene therapy, sickness prevention and forensic investigations and attracted substantial research efforts. Compared with traditional methods, electrochemical DNA sensors have shown potential applications owing to their relatively low cost, simplicity, high sensitivity, high specifity and ease to miniaturization.Guanine-rich nucleic acid sequence can fold into guanine quadruplex structure(G-quadruplex). Combined with hemin, the formed G-quadruplex-hemin is a kind of DNAzyme possessing peroxidase-like activity. G-quadruplex-hemin DNAzyme has been widely used as a signal amplification label in electrochemical sensor configuration. Based on the strategy of G-quadruplex-hemin signal amplification, we developed two highly sensitive electrochemical DNA biosensors. The details are summarized as follows:Firstly, a novel strategy was established for simultaneous electrochemical detection of dual target DNAs based on G-quadruplex-mediated amplification. A template DNA was firstly designed to be complementary to target DNA 1, target DNA 2, and part of signal DNA, which contains G-quadruplex forming sequence. In the presence of both target DNAs and signal DNA, these sequences hybridized with template DNA and were further ligated together to form a long strand under the catalysis of E. coli DNA ligase. After denaturation, the ligated sequence was dehybridized with template DNA and captured with a capture probe immobilized on the surface of gold electrode. With the help of hemin, G-quadrupelx-hemin complex can be formed on the surface of gold electrode which produced a remarkable electrochemical signal. Therefore, the proposed DNA sensor was used to simultaneously detect dual target DNAs with extremely high sensitivity. For target DNA 1 and target DNA 2, the detection limits of 100 fM and 7.4 fM were achieved respectively. The specificity of the sensor was verified by employing one-base mismatched and three-base mismatched sequences.Secondly, G-quadruplex-hemin amplification was coupled with nanomaterial-modified electrode to construct a label-free and ultrasensitive electrochemical DNA biosensor based on ExoIII assisted target-catalyzed recycling amplification strategy. In this biosensing system, two special hairpin structure probes termed P1 and P2 were designed. P1 probe contains three major domains I, II and III. Region I is a G-quadruplex-forming sequence which is blocked via hybridization with region III; and region II is the target DNA recognition domain. Region IV of P2 is complementary to the 3’ part of P1 and is caged via hybridization with region VI of P2 probe. The region V of P2 probe can be used as a secondary target to displace target DNA and can trigger the cleavage proces of Exo III. P1 probe was firstly immobilized on AuNPs-HDT-Au modified electrode by Au-S interaction. These hairpin structures of P1 and P2 can coexist in this system. In the presence of P2 and ExoIII, the target DNA can trigger two independent cycles of reactions: the hybridization reaction between P1 and target DNA and then through a branch migration process, P2 probe hybridize with the region III of P1, leading to the displacement of target DNA. The ExoIII can catalyze the cleavage reaction of P1-P2 duplex and releasing P2 as a secondary target DNA for the next cleavage cycle. With the presence of hemin, the region I, which is the only sequence remained on the electrode surface folded into G-quadruplex-hemin complexe, giving a remarkable elctrochemical signal. Using differential pulse voltametry, the detection limit of target DNA reached as low as 3.98 fM.