Design And Applications Of Signal Enhancement Strategies In DNA Aptamer-based Biosensing

With the increasing requirements on the sensitivity of biosensing system, various strategies have been devised to boost detection sensitivity of chemosensing and biosensing processes via the enhancement/amplification of sensing responses. The recognition element and the signal transducer are two core sections in a biosensing system. Various recognition elements have been developed in the past several decades, such as enzymes, lectins, antibodies, molecular imprinting and aptamers. Aptamer, which is single-stranded oligonucleotides with high affinity to a special target, is one of the emerging classes of versatile receptors. This study aims to the design and application of signal enhancement strategies in DNA aptamer-based biosensing. The main content is as follows:1. Because of its unique optical properties especially high extinction coefficient (3-5orders of magnitude higher than that of organic chromophores), gold nanoparticle (AuNP) was employed as the signal enhancement element and transducer to construct biosensing system for lead (II) detection. The sensing systems with two guanine-rich sequences (TBA (5’-GGT TGG TGT GGT TGG-3’) and PW17(5’-GGG TAG GGC GGG TTG GG-3’)) respectively as recognition elements were developed base on the principle that ummodified AuNP can distinguish unfolded ssDNA from folded ssDNA, such as G-quadruplex. The formation of G-quadruplexes by TBA-Pb2+and PW17-Pb2+were characterized by circular dichroism. The specificity of the sequence to Pb2+was analyzed by difference absorption spectrum. The AuNP of13nm synthesized using citrate reduction method was characterized by TEM and UV-vis spectroscopy. Experimental conditions, such as NaCl concentrations, ssDNA concentrations, aggregation time after the addition of salt were optimized. Results showed that a limit of detection of30nM can be easily obtained for Pb+detection. The PW17system was found to possess a much better performance for Pb2+detection than TBA system. In the same [Pb2+] range and ssDNA concentration, PW17system shows a larger LSPR response than TBA system with a relative smaller standard deviation.2. To investigate the difference of TBA system and PW17system in the performance of Pb2+sensing, the interaction between DNA G-quadruplexes and13nm gold nanoparticles (AuNPs) was studied. The adsorption of DNAs in G-quadruplex solutions onto AuNPs was observed in DNA-AuNP-based sensing system. The adsorption behavior was studied through monitoring of the localized surface plasmon resonance (LSPR) absorbance of13nm AuNPs at520and650nm (A650/A520) in the solutions of three widely studied guanine-rich sequences, TBA, PW17, and PSO (5’-GGG TTA GGG TTA GGG TTA GGG-3’). It was found that the degree of the adsorption of DNAs in Pb2+stabilized G-quadruplex solutions is up to93%after more than5h of incubation. Two interpretations, the adsorption of G-quadruplexes and the unfolding of G-quadruplexes in the presence of AuNPs, were proposed for these observations. To explore the possible explanation, the lead concentrations in the solutions containing G-quadruplex and AuNP were analyzed by inductively coupled plasma atomic emission spectrometer. The results showed that Pb2+had been released from the G-quadruplexes, which means the G-quadruplexes may be unfolded in the presence of AuNPs. The adsorption rate in PW17-Pb2+system was lower than that in TB A-Pb2+system, demonstrating that the G-quadruplex formed from PW17and Pb2+is more stable in the presence of AuNPs. This result can interpret the difference in their performance in Pb2+sensing. Similar results were also observed in PSO-K+system, which indicate that the potential unfolding of G-quadruplexes in the presence of AuNPs is a general phenomenon in DNA AuNP-based sensing system. This interaction between G-quadruplexes and AuNP demonstrated that long time incubation between DNAs and AuNPs would possibly make it unable to distinguish G-quadruplex from ssDNA.3. To avoid the adverse effect of unmodified AuNPs on the formation of folded structure of ssDNA, the thiolated-aptamer conjugated AuNPs sensing system was adopted for biosensors design. The newly designed AuNP functionalized with split aptamer was developed for the detection of adenosine triphosphate (ATP). The ATP aptamer was split into two parts with their5’prime or3’prime modified with thiol. Both the5’SH and3’SH modified strands for each split aptamer fragment were functionalized onto the same AuNP to construct double-functionalized AuNP-DNA conjugates. Thus, the split aptamer can be reassembled into intact folded structure in the presence of ATP molecule with two potential assembly types, which induces the assembly of AuNP-DNA conjugates. In this double-functionalized system, the traditional assembly type might facilitate another assembly type, which was found to give two-fold increase in LSPR response of AuNPs in the presence of ATP than the traditional assembly type, and improved the sensitivity for ATP detection. Time courses of the assemble processes with different assembly types, Mg2+concentrations, and aptamer fragments densities on AuNP were followed using the absorption ratio at650nm and520nm. A limit of detection of24μM with highly selectivity was determined which has greatly surpassed the traditional assembly type in ATP sensing.4. However, the double-functionalized AuNP-DNA system could only provide two-fold increase in LSPR response of AuNPs to target molecules than the traditional one. Therefore, a strand displacement reaction (SDR)-based catalytic cycle was employed to amplify signals. This system involves an entropy-driven catalytic cycle of two strand displacement reactions with five oligonucleotides, denoted as "Substrate-1","Fuel-1","Catalyst-1","Cl" and "C2", respectively. The "Catalyst-1" is an ATP aptamer catalyzing the SDRs to form the "Substrate-Fuel-1" duplexes. All the intermediates in the SDR processes have been identified by PAGE (polyacrylamide gel electrophoresis) analysis. Introduction of ATP into the SDR system will induce the "Catalyst-1" to form G-quadruplex conformation so as to inhibit the catalytic activity and cut down the formation of the "Substrate-Fuel-1" duplexes. Obviously, this target-inhibited catalytic cycle can be applied to an ATP sensing system. When the "Substrate-1" and "Fuel-1" oligonucleotides were labeled with a carboxyfluorescein (FAM) fluorophore and a4-([4-(dimethylamino)phenyl] azo)benzoic acid (DABCYL) quencher, this SDR catalytic system exhibits a "switch-on" response for ATP. Conditions for detecting ATP, such as the loading of the "catalyst", buffer concentration of Mg+and incubation temperature, have been optimized to afford a detection limit of50nM and a linear response up to1400nM of ATP. This target inhibited catalytic cycle provides an enzyme-free biosensing strategy with higher sensitivity than many aptamer-based biosensing systems and even some enzyme-based amplification systems.5. The problems, which exist in the above target inhibited catalytic cycle-based sensing system, are the multi-step operations and time-consuming detection process (8h). Therefore, a target triggered catalytic cycle was designed for biosensing. Besides the SDR-based catalytic cycle, this new system also contains a target-induced strand displacement process which releases the catalyst strand for the cycle from the ATP aptamer-"Catalyst-2" duplex. The sensing of ATP was achieved by labeling "Substrate-2" and "C4" strands with FAM and DABCYL respectively. The addition of ATP triggered the release of "Catalyst-2" so as to catalyze the SDR-based catalytic cycle. Then, the bounded "Substrate-2" and "C4" strands in "S-C-2" complex were separated in the presence of ATP and resuming the fluorescence. The intermediates in the system were analyzed by PAGE and the time course of the catalysis process was followed in the FAM and DABCYL labeled system. After optimizing the Mg2+concentration, this target triggered catalytic-based sensing system provided a more sensitive response (Limit of detection is20nM) to ATP than the target inhibited one. It also provides a faster detection process (less than1hour’s detection process) which overcomes the disadvantage in the target inhibited catalytic cycle-based system.