Biochemical Research On Aptamer Based Probes For Small Biomolecules And Metal Ions Determination

The aptamer, which was selected by an in vitro process known as SELEX (systematic evolution of ligands by exponential enrichment) is oligonucleotide sequence fragment that can selectively bind with target molecules. It provides properties of high specificity and affinity, easy to synthesise, easy to store and can bind almost any target existing in nature. It has been widely used in biological sensor for the detection of various biomolecules and heavy metal ions. This paper designed fluorescent aptamer sensor and electrochemical aptamer sensor based on the characteristics of aptamer probe for the detection of Hg2+ and adenosine. The detailed research and main results as follows:In Chapter 2, a simple, sensitive method based on fluorescence enhancement was established for determination of Hg2+by using T-rich DNA probes and intercalation dye SYBR Green I (SG). In the present of Hg2+, the single-strand T36 forms a double-stranded structure due to the formation of thymine-Hg2+-thymine base pairs (T-Hg2+-T). SG intercalates into the double-stranded DNA and produces intense fluorescence. The experimental results show that the intensity of the fluorescence emission increased upon the increasing concentration of Hg2+. It shows a good linear response of the fluorescence intensity to the Hg2+ concentration in the range of 500 nM～2.5 μM (I=0.4455C(nM)-86.96; R= 0.9999). The detection limit for Hg2+ is experimentally determined to be 373.6 nM.In Chapter 3, we herein report a label-free turn-on fluorescent sensor for mercuric ions, with up to a 10-fold increase in relative fluorescence signal after optimizations. Mercuric ions can selectively bind in between two DNA thymine bases and promote these T-T mismatches to form stable T-Hg2+-T structure, which makes the mercuric ions specific DNA form double-strand DNA. SYBR Green 1 was applied as reporter for the readout of the formation of the T-Hg2+-T base pairs. The background signal was reduced to zero with graphite oxide as the quencher at certain wavelengths. Under the optimized experimental conditions, it showed a good linear response of the fluorescence intensity to the Hg2+concentration in the range of 200 nM～2μM. The linear dependencies yielded regression equations of I=0.6444C(nM)-56.00, with the correlation coefficients of 0.9980. The detection limit for Hg2+ was experimentally determined to be 252.8 nM.In Chapter 4, a novel electrochemical aptamer sensing interface based on enzyme cascade amplification has been designed and constructed for detection of adenosine in this paper. In the presence of adenosine, the primer of the rolling circle amplification (RCA) was linked with the thiol-immobilized probe modified on gold electrode by E.coli DNA Ligase. The RCA was performed with phi 29 DNA polymerase to produce a long single strand, which was complementary to the circle template probe. Large amounts of gold nanoparticles labeled oligonucleotide probes were hybridized with the product of RCA. As a result, the impedance response of the sensing interface for recognition of adenosine was enhanced by the enzyme cascade amplification strategy. The proposed sensor possessed nice selectivity and reproducibility, and was applied to determination of adenosine samples with a linear range between 2.0μM and 100μM and a detection limit of 2.0μM. It can be seen that the strategy for design of the proposed sensing interface is expected to be used for any other targets detection as a general method.