Study On Electrochemical Biosensors Based On The Triplex-stranded DNA Molecular Switch

DNA molecular switch, assembled with nucleic acid, can reversible regulate the conformational through some target molecular and external environment such as temperature, pH and light. Hence, the DNA molecular switch was widely used in the research of electrochemical biosensor such as the single-strand DNA molecular switch or double-stranded DNA molecular switch-based electrochemical biosensor. Although these electrochemical biosensors have various advantages such as efficient, rapid, and simple, there were still some limitations which confine the development. For example, the covalent attachment of electroactive molecules at a location of a single-strand DNA molecular switch tends to significantly reduce the recognition probe's affinity and specificity toward the targets. Moreover, due to the flexible and randomness of single DNA, the poor stability of background signal should also be overcomed. Although the developed of double-stranded DNA molecule have been solved some shortages to some extent, there were still some shortcomings like the lack of reversible should be consideration. Apart from these strategies, triple-helix DNA molecular switch, which assembled for the basis of Watson Crick and Hoogsteen base pairings, have been successfully used for developing electrochemical biosensor. Compared to the single-strand DNA molecular switch or double-stranded DNA molecular switch, the triple-helix DNA molecular switch possesses some remark able features. First, there is no need to make any labeling of the recognition DNA, thereby keeping the binding affinity and specificity to target, and even enabling higher sensitivity. Second, the conformational could be reversible regulate by the concentration of ions and pH of solution. The mainly study as following:1. Aptamer/target binding-induced triple helix forming for signal-on electrochemical biosensingOwing to its diversified structures, high affinity, and specificity for binding a wide range of non-nucleic acid targets, aptamer is a useful molecular recognition tool for the design of various biosensors. Herein, we report a new signal-on electrochemical biosensing platform which is based on an aptamer/target binding-induced strand displacement and triple-helix forming. The biosensing platform is composed of a signal transduction probe (STP) modified with a methylene blue (MB) and a sulfhydryl group, a triplex-forming oligonucleotides probe (TFO) and a target specific aptamer probe (Apt). Through hybridization with the TFO probe and the Apt probe, respectively, the self-assembled STP on Au electrode via Au-S bonding keeps its rigid structure. The MB on the STP is distal to the Au electrode surface. It is eT off state. Target binding releases the Apt probe and liberates the end of the MB tagged STP to fold back and form a triplex-helix structure with TFO (STP/TFO/STP), allowing MB to approach the Au electrode surface and generating measurable electrochemical signals (eT on). As test for the feasibility and universality of this signal-on electrochemical biosensing platform, two aptamers which bind to adenosine triphosphate (ATP) and human a-thrombin (Tmb), respectively, are selected as models. The detection limit of ATP was 7.2 nM, whereas the detection limit of Tmb was 0.86 nM.2. Target binding-induced polymerase signal amplification for electrochemical detection of nucleic acids via a triple-helix molecular switchIn this method, the triple-helix molecular switch consists of a central target specific nucleic acids sequence flanked by two arm segments (the DNA was defind as HP) which was riched in pyrimidine base and a dual-labeled oligonucleotide (the DNA was defind as A) designed as a hairpin-shaped structure and labeled with sulfhydryl group and methylene blue (MB) at the 5'and 3'end, respectively.The triple-helix molecular switch was firstly modified on the surface of gold electrod through Au-S bonding. Owning its rigid structure, the MB was distal to the Au electrode surface, and then generating a relatively low electrochemical signal (eT off). With the addition of the target DNA, the hybridization of target DNA with the HP would displace the HP from the triple-helix molecular switch. As a reulst, the A DNA could subsequently form a hairpin-shaped configuration, allowing the MB to easily collide with the electrode surface and generating an enhanced electrochemical signal (eT on). At the same time, the primer DNA can recognize the rleased HP and initiate the polymerase chain reaction to produce massive expension sequences, which can hybrided with HP and displace the target DNA. The displaced target DNA was further reacted to triple-helix molecular switch, and an amplified signal was then obtained. The detection limit of target DNA was 30 pM.