Study On The Construction And Application Of Aptamer Sensing Methods Based On The Triplex-stranded DNA Molecular Switch

The conformational transition of aptamers by the induction of the target was thekey of the aptamer-based sensor to achieve a shifty signal. There were many probesdesigned based on this theory in the biosensor. Such as the single-stranded DNAaptamer probes, the double-stranded aptamer probes or molecular beacons, however,these stratagies had some flaws. For example, in the sensor applications thecombination of the label-free aptamer with electrochemical impedance methods,electrostatic adsorption of cationic polyelectrolyte or embedded indicator, thesestratagies although well maintained the affinity and specificity of aptamer and hadadvantages about quick, easy, cheap, etc., but they were difficult to obtain highsensitivity and combine the methods of remarkable signal. And in the sensorapplications using the labeled aptamer, not only the specificity and affinity of theaptamer was affected, however, such single “signal on” or “signal off” sensingmethods suffered from limited signaling capacity, in which only a maximum of100%signal suppression can be attained under any experimental conditions. And thetriplex-stranded DNA was formed based on Watson-Crick and Hoogsteen basepairings. In this configuration, a single-stranded oligonucleotide by hydrogen bondedinto the major groove of double-stranded DNA structure, the parallel or anti-paralleltogether to form. Based on these, in this paper, the unique configuration oftriple-stranded DNA was used to design the aptamer-based sensing analysis methods.The main works were summarized as follows:1. A signal on aptamer-based electrochemical sensing platform usingtriple-stranded DNA molecular switch.We developed an aptamer-based electrochemical sensing platform. Atriplex-stranded molecular switch was employed in this electrochemical sensingplatform. The triple-stranded molecular switch called APT-THDNA contained twoportions: a label-free target specific aptamer sequence (APT) flanked by two armsegments, as a recognition probe; a signal transduction probe (STP) designed as ahairpin-shaped structure and labeled with methylene blue (MB) and sulfydryl group atboth ends. The two arm segments of the APT could hybridize with the loop sequenceof STP and form the APT-THDNA. By Au–S bonding, the APT-THDNA was thenmodified on the electrode. When target was absent, the APT-THDNA kept intact on the electrode with STP in its “open” configuration. The MB was far from theelectrode. It was “eT off” state. In the presence of target, the APT recognized thetarget. Subsequently, the APT was released from the Apt-THDNA, leading to newsignal readout with MB closing to the surface of gold electrodes. It was “eT on” state.Human α-thrombin (Tmb) was used as the first model to show the feasibility andapplicability of electrochemical sensing platform, and the limition of detection was4.5nM. Further more, the universality of the sensing platform was demonstrated byvirtue of altering the Tmb aptamer sequence to adenosine triphosphate (ATP) aptamersequence, and the limition of detection was60nM. The results demonstrated that thissensing platform was selective and universal.2. Sensitive triplex-stranded DNA molecular swith coupling double signal“signal-on” and “signal-off” for fluorescent sensing method to detect ATP.Based on an aptamer, we designed of a triplex-stranded switch by the inductionof target coupling double signal “signal-on’’ and“signal-off’’ to develop a highlysensitive fluorescence aptasensor analytical method. The aptamer-based double signalfluorescent probe (ATFS) contained three portions: a probe labeled with a fluorophore(FAM), a probe labeled with tetramethylrhodamine (TMA), and a probe labeled with aquencher group (BHQ-2). And the both ends of BHQ-2probe hybridizated with theFAM probe and the TMA probe. In the absence of ATP, the fluorescence of FAM wasquenched by BHQ-2probe. The fluorescence of the TMA was kept. In the presence ofATP, however, the fluorescence of FAM was recovered due to the competition of ATPwith its aptamer. At the same time, the fluorescence of TMA is quenched andweakened by the formation of triplex-stranded configuration. When the total value ofthe two signals was used as the response signal for determination of ATP, we couldachieve a more sensitive detection of the target results. Without any amplificationstrategy, this fluorescence detection method improved detection signal, and had anon-radioactive, simple preparation. The method detection limit of ATP as low as1nM. Slight sequence modifications of the probe, the method can also be used to detectother target.