Building Novel Adenosine And Hg (II) Sensors With Functional DNA Based Allosteric Assembly

Two novel structured sensor assemblies for adenosine and Hg (II) were successfully built based on functional DNA molecules and were well characterized. These new sensors all exhibited satisfying sensitivities and excellent specificities against various analogous species. In addition, ferrocene carboxylic acid derivated functional small molecule compounds were synthesized through N-hydroxysuccinimide ester activated amidation reactions. A DNA-based signal-on type adenosine electrochemical sensor was also designed and investigated by preliminary experiments. More details are given below:Split hemin-binding G-quadruplex DNAzyme halves and an anti-adenosine aptamer was employed to construct a homogeneous assay for optical sensing of adenosine. In the presence of potassium ions, the two DNAzyme halves would cooperatively transform into an intact G-quadruplex after capturing a hemin molecule, which then became a hemin-intercalated DNAzyme with peroxidase-like activities for the catalytic oxidation of ABTS by H2O2. The oxidation reaction could generate an absorbance change of a assay solution due to the accumulation of ABTS?+ (a cationic radical product during the oxidation of ABTS (2, 2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid)), which was then taken as the output of the sensor. The introduction of adenosine would trigger an intramolecular conformational change of the sensor structure due to the binding of adenosine to its aptamer part embedded in the sensor module, which then prohibited the correct formation of the G-quadruplex-hemin complex and lowered the overall peroxidase activity of the sensor assembly with decreased optical absorbance output of the assay. Thanks to the highly specific affinity of adenosine to its aptamer, the adenosine sensor was highly selective against various possible interferences including guanosine, cytodine and uridine. This homogeneous, easy-to-built, low cost and colorimetric sensor offered a satisfying detection limit of 6μM for adenosine under still not fully optimized detection conditions.As a re-engineered version of the adenosine sensor, a label-free colorimetric Hg (II) sensor was also designed by rationally integrating a mercury specific T (thymine) -rich DNA segment, two split G-quadruplex halves and a rigid duplex spacer in a whole. Similarly, the split G-quadruplex enzyme served as a signal transduction module in this sensor. Interaction of Hg(II) with the T-rich DNA domain within the sensor induced the formation of a hairpin structure stabilized by multiple non-canonical T-Hg²⁺-T basepairings, which, similar to the adenosine sensor, finally led to a decreased colorimetric output of the sensor assay. This sensor had a high sensitivity for Hg(II) with a limit of detection (LOD) of 4.5 nM (0.9 ppb), which is higher than or comparable with previously reported Hg²⁺ assays and lower than the toxic level defined by U.S. Environmental Protection Agency (EPA) in drinkable water (10 nM). It also had very high specificity to Hg(II), and up to 20μM of 10 other metal ions did not show any interferences with the detections under suitable conditions. This simple, modular and easy-to-build Hg²⁺ assay required no synthetic efforts such as chemical labelings or nanoparticle preparations and conjugations, which further lowered its detection cost and increased system stability.Ferrocene derivatives of some amino compounds were synthesized. Some of the products were isolated on a silica column and characterized. Ferrocene functionalized cystamine was assembled on a gold electrode and its electrochemical behaviors were investigated. In addition, a DNA-based signal-on electrochemical sensor of adenosine was designed and some preliminary experiments were carried out.