Construction Of Electrochemical Aptasensor Based On Exonuclease-assisted Amplification And Its Analytical Applications

Protein is an important macromolecule material that constitutes the cells and tissues of humans. It is the material basis which plays important roles such as transport, catalysis, immunization and adjustment function in the various life activities. Therefore, the realization of high sensitive detection of protein is significant in disease diagnosis and clinical treatment. Thrombin is a kind of serine protease which function as terminal enzyme of coagulation system in human body. It plays a key role in the blood coagulation cascade. The concentration and activity of thrombin is one of the important indicators to measure blood clotting mechanism, it is an important reference in early diagnosis of disease. Heavy metal ions are highly toxic materials which have carcinogenic and teratogenic effect. Heavy metal pollution has caused serious damage to the ecological environment and human health. Hg2+ is bioaccumulative pollutant with highly toxicity, which seriously affects the normal metabolism of cells, the central nervous system and digestive system. Aptamers are also celled "chemical antibodies", compared with antibody, it has a series of advantages such as high affinity and good specificity, easy to be modified and labeled, denaturation and renaturation is reversible and so on. It can specific recognize targets such as proteins, small molecules and cells. The employment of aptamer in electrochemical sensor combines the advantage of them, providing a new method for biochemical analysis.In this paper, by using exonuclease-assisted target recycling and hybridization chain reaction amplification strategy for signal amplification, we developed electrochemical aptasensor for the detection of protein and heavy metal ion based on specificity recognition between aptamer and target. The major content of the paper includes the following aspects:(1) In this work, a dual-signaling electrochemical aptasensor based on exonuclease-catalyzed target recycling was developed for thrombin detection. The proposed aptasensor coupled "signal-on" and "signal-off’ strategies. As to the construction of the aptasensor, ferrocene (Fc) labeled thrombin binding aptamer (TBA) could perfectly hybridize with the methylene blue (MB) modified thiolated capture DNA to form double-stranded structure, hence emerged two different electrochemical signals. In the presence of thrombin, TBA could form a G-quadruplex structure with thrombin, leading to the dissociation of TBA from the duplex DNA and capture DNA formed hairpin structure. RecJf exonuclease could selectively digest single-stranded TBA in G-quadruplex structure and released thrombin to realize target recycling. As a consequence, the electrochemical signal of MB enhanced significantly, which realized "signal on" strategy, meanwhile, the deoxidization peak current of Fc decreased distinctly, which realized "signal off’ strategy. The employment of RecJf exonuclease and superposition of two signals significantly improved the sensitivity of the aptasensor. In this way, an aptasensor with high sensitivity, good stability and selectivity for quantitative detection of thrombin was constructed, which exhibited a good linear range from 5 pM to 50 nM with a detection limit of 0.9 pM (defined as S/N= 3). In addition, this designed strategy could be applied to the detection of other proteins and small molecules.(2) In this work, a novel electrochemical aptasensor was developed for Hg2+ detection based on exonuclease-assisted target recycling and hybridization chain reaction (HCR) dual signal amplification strategy. The presence of Hg2+ induced the T-rich DNA partly folded into duplex-like structure via the Hg2+ mediated T-Hg2+-T interaction, which triggered the activity of exonuclease III (Exo III). Exo III selectively digested the double-strand DNA containing multiple T-Hg2+-T base pairs from its 3’-end, the released Hg2+ participated analyte recycle. With each digestion cycle, a digestion product (help DNA) was obtained, which acted as a linkage between the capture DNA and auxiliary DNA. The presence of help DNA and two auxiliary DNA collectively facilitated successful HCR process. [Ru(NH3)6]3+was used as redox indicator, which electrostatically bound to the double-strand DNA and produced an electrochemical signal. Exo Ⅲ-assisted target recycling and HCR dual amplification significantly improved the sensitivity for Hg2+, demonstrating a good linear range from 0.2 pM to 35 nM with a detection limit of 0.12 pM (S/N= 3). Furthermore, the proposed aptasensor had a promising potential for the application of Hg2+ detection in real aquatic sample analysis.(3) In this work, a simple and sensitive electrochemical aptasensor of Hg2+ was developed, which utilized exonuclease III (Exo III) and hybridization chain reaction (HCR) to amplify electrochemical signal. T-rich capture probe was self-assembled on the gold electrode, with the presence of Hg2+, two adjacent capture probe (CP) can capture Hg2+, led to the formation of double-strand DNA containing T-Hg2+-T structure. The T-Hg2+-T interaction triggered the activity of Exo III, the T-Hg2+-T structure was digested by Exo Ⅲ and Hg2+ was released for target recycling. The remainder CP and two auxiliary DNA collectively led to successful HCR process, which formed long double-stranded DNA and produced large amount of adsorption sites for the electroactive molecules ([Ru(NH3)6]3+), which resulted in significant electrochemical signal output. Exo Ⅲ-assisted target recycling and HCR dual amplification strategy significantly improved the sensitivity for Hg2+ with the liner range of 0.25 pM-100 nM and a detection limit of 0.14 pM (S/N= 3). The strategy could be extended to the detection of other small including small molecule and proteins.