DNA Amplification-Based Aptasensor For Heavy Metal Ions Detection In Environment

Because of the harm to environment with enrichment, long-term, universality and seriousness, the pollution of heavy metal ions has attracted more and more attention. And thus developing timely, simple, accurate, sensitive and selective methods for the detection of heavy metal ions in environment is the base, premise even the key point to prevention, controlling and treatment of heavy metal ions pollution. Compared with the traditional methods for detection of heavy metal ions, electrochemical and electrochemiluminescence aptasensor possess both the advantages of electrochemical and electrogenerated chemiluminescence analysis, as well as the advantages of aptamer, including fast response, simple operation, strong specificity, high sensitivity and so on, which provide great feasibility for the detection of heavy metal ions and would become one of the most promising detection method. At present, utilizing the signal changes that only caused by aptamer conformational change for target analysis is limited by the sensitivity. Therefore, in order to further improve the sensitivity of aptasensor and realize the trace detection of heavy metal ions, many signal amplification strategies need to be used here. Hybridization chain reaction (HCR) is a kind of newly DNA-based amplification, which causes a reaction at room temperature. And the auxiliary for HCR also need no control of the temperature change and other enzymes, which only relies on the complementary pairing by a pair of cross complementary oligonucleotide probe. The HCR provides a potential possibility to construct simple and sensitive electrochemical and electrochemiluminescence aptasensor for detection of heavy metal ions. Based on the above observation, this thesis utilized the HCR as basic amplification strategy, and combined with the mimic enzyme catalyzed signal amplification to construct a series of electrochemical and electrochemiluminescent aptasensor, aiming to construct simple, reliable, rapid, sensitive and selective aptasensors for the detection of one or more heavy metal ions on the same sensitive interface. Additionally, the recovery of standard addition or actual sample detection was employed to verify the feasibility and reliability of proposed aptasensors. This work focuses on the construction of sensitive interface, the researching of specific binding between heavy metal ions and base, and the development of novel electrochemical signal amplificatory strategy. The detail contents are as follows:(1) hemin/G-quadruplex is formed by interlacing hemin into a single-stranded guanine-rich nucleic acid and could hinder the electron transportation. Based on this principle, we constructed an impedimetric DNA biosensor for silver ions (Ag+) detection with [Fe(CN)6]4-/3- as redox probe and HCR induced hemin/G-quadruplex nanowire as enhanced label. In the present of target Ag+, Ag+ interacted with cytosine-cytosine (C-C) mismatch to form the stable C-Ag+-C complex with the aim of immobilizing the primer DNA on electrode, which thus triggered the HCR to form inert hemin/G-quadruplex nanowire with an amplified electrochemical impedance spectroscopy (EIS) signal. According to the increased EIS signal, the DNA biosensor showed a high sensitivity with the concentration range spanning from 0.1 nM to 100 mM and a detection limit of 0.05 nM. Compared to other analogously reported DNA based biosensors, the proposed biosensor showed a relatively higher sensitivity and wider detection range, which further demonstrated hemin/G-quadruplex nanowires could effectively hinder electron transfer for signal enhancing. In addition, the feasibility of the proposed AgT biosensor was investigated via standard addition methods in river water (collected from Yangtze Gorges, China), and the satisfied recovery ranged from 90.1% to 108.0%, providing a promising tool for Ag+ detection with enough precision and accuracy even in real environmental sample matrices.(2) Hemin/G-quadruplex could not only hinder the electron transportation, but also act as HRP-mimicking DNAzyme to show great catalytic activity for H2O2 hydrolysis. Actually, the electrochemiluminescence (ECL)-based biosensor may be more sensitive than electrochemical-based biosensor. Hence, we constructed a novel ECL DNA biosensor for Ag+ sensitive detection based on the in situ produced hemin/G-quadruplex DNAzyme nanowires as enhancer with the aid of HCR. Firstly, C-rich nucleic acid capture probe were immobilized onto depAu decorated glass carbon electrode surface. Following that, the carried capture probe propagated the chain hybridization with primer to trigger HCR for the formation of hemin/G-quadruplex DNAzyme nanowires. In the electrolyte of PBS (pH 7.4) containing luminol and H2O2, hemin/G-quadruplex DNAzyme exhibited excellent catalysis towards H2O2 to generate amounts of ROSs. Coupling HCR with hemin/G-quadruplex DNAzyme, the proposed ECL biosensor exhibited an enhanced ECL response for detection of Ag+ In the present of target AgT Ag+ interacted with adjacent C-C mismatch to form the stable C-Ag+-C complex with the aim of releasing hemin/G-quadruplex DNAzyme nanowires from electrode surface, which thus the decreased hemin/G-quadruplex nanowire leading a decreased ECL signal for AgT detection.. The proposed ECL biosensor presented predominate selectivity and high sensibility for determination of Ag in the range from 2 pM to 20 μM with a detection limit of 0.67 pM. Compared with our previous proposed impedimetric DNA biosensor, this biosensor presented a wider liner range and lower detection limit, indicating the designed biosensor could be extended toward the on-site monitoring of the other metal ions or trace pollutants in environmental matrices by using different DNA or aptamer probes.(3) Manganese(Ⅲ) meso-tetrakis (4-N-methylpyridyl)-porphyrin (MnTMPyP) is a typical manganese porphyrin that contains manganese as the central metal atom. It can interact with both the AT and GC base pairs of double-strand DNA (dsDNA) by groove binding and exhibit superior peroxidase-like activity. Compared with the commonly used hemin/G-quadruplex, a complex with iron porphyrin hemin functions as a G-rich, single-strand DNA and shows peroxidase-like activity; the MnTMPyP-dsDNA does not require a specific sequence, and the amount of MnTMPyP loaded in the dsDNA scaffold is relatively much larger, which provides a new avenue for the preparation of simple and sensitive electrochemical DNA biosensors with high catalytic efficiency. Herein, based on the MnTMPyP and electron mediator thionine (Thi) co-decorated DNA nanowires, we prepared a simple and sensitive electrochemical DNA biosensor for the trace detection of toxic mercury (II) ion (Hg2+). The T-rich capture DNA was first assembled on the nano-Au modified glass carbon electrode through Au-S bond. Through specific base-pairing, the primer DNA was thus modified on the electrode surface to trigger the hybridization chain reaction (HCR) with the aim of forming the MnTMPyP and Thi co-decorated DNA nanowires. In the electrolyte containing H2O2, the MnTMPyP loaded in the DNA nanowires showed superior peroxidase-like activity, which quickly electrocatalyzed the reduction of H2O2, promoting the redox reaction of Thi with a dramatically amplified electrochemical signal. However, in the presence of target Hg2+, Hg2+-mediated thymine base pairs (T-Hg2+-T) were formed between the two neighboring T-rich capture DNA, which resulted in the release of MnTMPyP and Thi co-decorated DNA nanowires from electrode surface, providing a reduced readout signal for the quantitative electrochemical detection of Hg2+. The proposed biosensor exhibited excellent sensitivity and the analyte concentrations in the ranges from 5 pM to 50μM with a detection limit of 2.5 pM and also had high selectivity toward Hg2+(4) Most of biosensors focus on single metal ion assay, while Hg2+ and Ag+ are usually co-present in water. Consequently, designing a method for the simultaneous detection of Hg2+ and Ag+ on one-spot is necessary and meaningful since it can shorten analytical time, decrease detection cost and improve the test efficiency. Here, we develop a sensitive and selective strategy method for the simultaneous detection of Hg+ and Ag+ by target-induced conformational switch and DNA concatemers amplification protocol. In this approach, two dsDNA strands, one is thiolate-labeled C-rich oligonucleotide (C1) and its partially complementary DNA (P1), the other is thiolate-labeled T-rich oligonucleotide (C2) and its partially complementary DNA (P2), were firstly self-assembled on a gold nanoparticles modified electrode. And then, when four auxiliary DNA strands, anthraquinone-2-carboxylic acid (AQ)-labeled LS1 and LS2, thionine (Thi)-labeled LS3 and LS4, were introduced, extended dsDNA polymers with lots of AQ moieties and Thi moieties complexes could form on the electrode surface, which were employed P1 and P2 as primers for the concatemer reaction. In the presence of Ag+ and Hg2+, C1 and C2 on electrode via the insertion approach can fold into hairpin structures, resulting in the release of the redox-tagged DNA concatemers from the electrode surface with a substantially decreased redox current, which could be used to detect the target. Based on the electrochemical out puts of the surface-captured AQ and Thi complex at distinct potentials with different sizes, the identities and concentrations of Ag+ and Hg2+ could be identified respectively. As the experiment results indicated, the calibration plots showed a good linear relationship between the peak currents and the analyte concentrations in the ranges from 0.01 nM to 5 μM with a detection limit of 2 pM for Ag+, and 0.05 nM to 0.05 mM with a detection limit of 7.5 pM for Hg2+, respectively.