| In view of the advantages such as good specificity and stability, easy synthesis and modification, and flexible output signal mechanism, aptamers have become important molecular probes and play vital roles in analytical chemistry. In order to develop a highly sensitive aptasensor, a great deal of signal amplification strategies, such as enzyme-assisted target recycling and hybridization chain reaction have been widely used for highly sensitive detection of nucleic acids, ions, proteins, small molecules and so on. Since Ono and Togashi affirmed that a pair of thymine(T) as ligand can specifically recognize free Hg2+,there are numerious reports of Hg2+ detection based on T-Hg2+-T complex. Recently, it has been authenticated that catalysis activity of Exo Ш can be activated by T-Hg2+-T base pairing by Hsing et al. Thus, Exo Ш-assisted target recycling tactic has been employed to develop signal-amplified sensor platform for Hg2+ detection with preferable selectivity and sensitivity. By taking advantage of T-Hg2+-T structure and abbility of the Exo Ш digestion, sensitive and selective sensors were developed. In this thesis, we design several novel aptasensors to improve the detection sensitivity of DNA and heavy metal ions based on multiply signal amplification containing enzyme-assisted target recycling, DNAzyme catalysis, hybridization chain reaction and G-wires. The details are summarized as follows: 1. Ultrasensitive and selective signal-on electrochemical DNA detection via exonuclease III catalysis and hybridization chain reaction amplificationUltrasensitive and selective detection of nucleic acids plays an important role due to its broad applications in biological studies, forensic evidence and early screening of cancers. A novel, ultrasensitive, and selective platform was constructed for electrochemical detection of DNA by integrating of exonuclease III(Exo-III) assisted target recycling and hybridization chain reaction(HCR) as the dual signal amplification strategy. The hairpin capture probe DNA(C-DNA) with an Exo-III 3′ overhang end was self-assembled on a gold electrode. In the presence of target DNA(T-DNA), C-DNA hybridized with the T‐DNA to form a duplex region, exposing its 5′ complementary sequence(initiator). Exo-III was applied to selectively digest duplex region from its 3-hydroxyl termini until the duplex was fully consumed, leaving the remnant initiator. The intact T-DNA spontaneously dissociated from the structure and then initiated the next hybridization process as a result of catalysis of the Exo-III. HCR event was triggered by the initiator and two hairpin helper signal probes labeled with methylene blue, facilitating the polymerization of oligonucleotides into a long nicked dsDNA molecule. The numerous exposed remnant initiators can trigger more HCR events. Because of the integration of dual signal amplification and the specific HCR process reaction, the resultant sensor showed a high sensitivity for the detection of the target DNA in a linear range from 1.0 fM to 1.0 nM(r = 0.9953), and a detection limit as low as 0.2 fM. The specificity of the proposed scheme was investigated by exposing the sensor to four kinds of DNA sequences, including the T-DNA, single-mismatched DNA, three-mismatched DNA and non-complementary DNA at the same concentration. The results indicated that the proposed approach showed good specificity for detecting mismatched DNA. The proposed dual signal amplification strategy provides a powerful tool for detecting different sequences of target DNA by changing the sequence of capture probe and signal probes, holding a great potential for early diagnosis in gene-related diseases. 2. Label-free colorimetric detection of Hg2+ based on Hg2+-triggered exonuclease III-assisted target recycling and DNAzyme amplificationThis work reported a label-free colorimetric assay for sensitive detection of Hg2+ based on Hg2+-triggered hairpin DNA probe(H-DNA) termini-binding and exonuclease Ш(Exo Ш)-assisted target recycling, as well as G-quadruplex/hemin(DNAzyme) signal amplification. The specific binding of free Hg2+ with the thymine-thymine(T-T) mismatches termini of H-DNA could immediately trigger the Exo Ш digestion, and then set free G-quadruplex segments and Hg2+. The Exo Ш impellent recycling of ultratrace Hg2+ produced numerous G-quadruplexes. The corresponding DNAzymes catalyzed efficiently the H2O2-mediated oxidation of the ABTS2- to the colored product in the presence of hemin. Using the color change as the output signal, and the Exo Ш-aided Hg2+ recycling and DNAzyme as the signal amplifier, the proposed sensing strategy implemented in aqueous solution had a definite advantage in naked-eye detection of Hg2+ as low as 1.0 nM. The ultrasensitive assay system successfully achieved visual detection of Hg2+, and was portable and low-cost. The reultant aptasensor was successfully applied to colorimetric detection of Hg2+ in laboratory tap water and Jialing river water samples. Satisfactory values between 95.2% and 106% were obtained for recovery experiments.3. Ultrasensitive label-free resonance Rayleigh scattering aptasensor for Hg2+ detection using Hg2+-triggered exonuclease III-assisted target recycling and growth of G-wires forsignal amplificationThe RRS intensities were significantly increased when Mg2+ was added to the parallel-stranded G-quadruplex(c-myc) solution because Mg2+ can trigger the formation of guanine nanowires(G-wires) superstructure polymer. However, the RRS spectrum and intensity could not be changed by adding Mg2+ to single strand, duplex strands, triplex strands, i-motif or antiparallel-stranded G-quadruplex as it cannot change the structure of these oligomers. Thus, a novel signal-on and label-free resonance Rayleigh scattering(RRS) aptasensor was constructed for detection of Hg2+ based on Hg2+-triggered exonuclease III-assisted target recycling and growth of G-quadruplex nanowires(G-wires) for signal amplification. The hairpin DNA(H-DNA) was wisely designed with thymine-rich recognition termini and a G-quadruplex sequence in the loop. The H-DNA was employed as a signal probe for specially recognizing trace Hg2+ by a stable T-Hg2+-T structure, which automatically triggered Exonuclease III(Exo-III) digestion to recycle Hg2+ and liberate the G-quadruplex sequence. The free G-quadruplex sequences were self-assembled into guanine nanowire(G-wire) superstructure in the presence of Mg2+, which was demonstrated by gel electrophoresis. The RRS intensity was dramatically amplified by the resultant G-wires, and the maximum RRS signal at 370 nm was linear with the logarithm of Hg2+ concentration in the range of 50.0 pM to 500.0 nM(R = 0.9957). Selectivity experiments revealed that the as-prepared RRS sensor was specific for Hg2+, even coexisting with high concentrations of other metal ions(Zn2+, Mn2+, Fe3+, Ca2+, Co2+, and Cu2+). This optical aptasensor was successfully applied to identify Hg2+ in laboratory tap water and river water samples, and satisfactory values between 94.0 and 108.0% were obtained for the recovery experiments.4. A Label-free resonance Rayleigh scattering aptasensor for Pb2+ detection based on dual signal amplification of Pb2+-dependent DNAzyme and G-wiresA resonance Rayleigh scattering(RRS) sensor was constructed for detection of Pb2+ based on Pb2+-dependent DNAzyme enzymatic target recycling and formation of G-quadruplex nanowires(G-wires) for signal amplification. A hairpin DNA labeled with rA in the loop was designed for Pb2+ cleavage recognition, and part of G-rich sequence was designed in the stem of hairpin DNA. The H-DNA was cleavage by Pb2+ and DNAzyme to yield parallel stranded G-quadruplexes which further were induced into G-wires superstructure in the presence of Mg2+. The G-wires were demonstrated by gel electrophoresis and AFM. The RRS intensity was dramatically amplified by the resultant G-wires, and the maximum RRS signal at 380 nm was linear with logarithm of Pb2+ concentration in the range of 2.0 nM to 5.0 μM(R = 0.9970). Selectivity experiments revealed that the as-prepared RRS aptasensor was specific for Pb2+ even coexisting with high concentrations of other metal ions. This optical aptasensor was successfully applied to determination of Pb2+ in laboratory tap water and river water samples. With excellent sensitivity and selectivity, the proposed RRS aptasensor was potentially suitable for not only routine detection of Pb2+, but also a universal DNAzyme-based sensing platform for sensitive detection of various metal ions targets just by changing the recognition sequences of the probe and DNAzyme. |
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