Homogeneous Electrochemiluminescence Aptamer-based Detection Of Thrombin

Nucleic acid and protein are two kinds of most important molecules in cells. The interaction between proteins and nucleic acids is one of the key issues in molecular biology. The success of Human Genome Project resulted in a lot of genes found and located. The function problem of the Human Genome is a research hotspot. The majority of the final products of gene are the corresponding proteins, and thus recognition the function of gene needs to study the corresponding proteins, in which many new methods have introduced to study the nucleic acid /protein binding interactions, such as aptamer-based methods. Detecting and measuring levels of proteins is one of the most fundamental and most often performed experiments in biomedical research.The electrogenetated chemiluminescence (ECL) reaction was initiated by an electrochemical reaction at electrode surface. Such an electro-initiation approach introduces many distinct advantages such as high sensitivity, wide range, rapid and convenient operation. Immunoassay is an analytical methodology that has been widely employed in clinical, pharmaceutical and biochemical fields due to its high specificity of antibody/antigen. Our group has developed a series of homogeneous ECL immunoassay methods for the determination of digoxin antibody and digoxin hapten. However, the drawbacks of the antibody associated with its production, stability and manipulation have prompted researchers to seek alternatives. Aptamer taken as a new type of molecular recognition elements has received much attention in design of bioaffinity sensors and assay in recent years. Aptamer selected from combinatorial libraries using systemic evolution of ligands by exponential enrichment (SELEX) is oligonucleotide (DNA or RNA), which can bind with high affinity and specificity to a wide range of target molecules including drugs, proteins and other organic or inorganic molecules. Unlike antibody, aptamer can be synthesized chemically and selected by the SELEXprocess. Additionally, aptamer can undergo ligand-dependent conformational changes and offer a long-term stability, target versatility, and convenient regeneration. Because of its specific abilities, it would be extremely useful to make aptamer-based bioanalysis for the determination of protein and small molecular substances.The aim of the present work was to develop a simple and highly sensitive homogeneous ECL method combination of a highly binding aptamer to protein analyte for the detection of target protein-thrombin. Ruthenium bis(2,2'-bipyridine) (2,2'-bipyridine-4,4'-dicarboxylic acid) N-hydroxysuccinimide ester (Ru(bpy)₂(dcbpy) NHS) was used as a ECL label. Research work in this thesis is financially supported by the National Natural Science Foundation of China (Grant No. 20775046, No. 20375025). This thesis includes two parts. First part, chapter 1, involves general introduction of aptamer and research development, and the purpose of this research work. The second part including two chapters presents research work.In the first subunit of chapter 2, a novel homogeneous electrogenerated chemiluminescence (ECL) method for the determination of thrombin was developed employing an ECL probe consisting of thrombin aptamer as a recognition molecule and tris (2, 2'-bipyridyl) ruthenium derivatives served as an ECL tag. ECL measurement was performed at a constant potential of 1.20 V in 0.10 mol/L TPA and record the signal of ECL emission. It was found that in the absence of thrombin, a strong ECL emission is electrochemically generated from Ru(bpy)₂(dcbpy)NHS-labeled aptamer. In the presence of thrombin, formation of the complex of thrombin binding to ruthenium complex-tagged G-quadraplex conformation of the thrombin aptamer, thus the ECL emission markedly decreased due to the steric hindrance allowed to remove the tag of Ru(bpy)₂(dcbpy)NHS away from the electrode surface and thus the ECL signal was dropped off. The proposed method described herein has advantages such as not only simplify, rapidness and efficient sensitivity but also no need to immobilized aptamer on the electrodes by thiol-self-assembly. The changes of integrated ECL intensity had a linear relationship with logarithm of concentrations of thrombin in the range of 5.0×10^-10～1.0×10^-7mol/L and the linear regression equation wasΔI_ECL = - 219 + 732 lgC(unit of C is 10^-10 mol/L, r=0.9963). The detection limit for thrombin was 1.2×10^-10 mol/L (S/N = 3) and the relative standard derivation for 5.0×10^-9 mol/L was 2.7% (n = 7).In the second subunit of chapter 2, a sensitive electrogenerated chemiluminescence (ECL) aptamer-based detection of thrombin coupled to magnetic beads by using single-walled carbon-nanotubes (SWNT) carrying a large number of ruthenium bis(2,2'-bipyridine) (2,2'-bipyridine-4,4'-dicarboxylic acid bis[(2-aminoethane) amide]) (ruthenium complex) tags has been developed. This aptamer-based sandwich assay for ECL detection of thrombin was designed in three steps, first, aptamer-functionalized magnetic beads and second binding with target thrombin, then further binding with the ECL probe to form sandwich complex. By monitoring the ECL intensity, the binding event was detected between the aptamer and thrombin for thrombin quantization. The ECL intensity had a linear relationship with logarithm of concentrations of thrombin in the range of 1.0×10^-11～5.0×10^-9 mol/L, and the linear regression equation was I_ECL = 676.98lgC_thrombin+ 7488.5 (unit of C is 10^-11mol/L, r=0.9921). A detection limit is 1.3×10^-12 mol/L. The relative standard derivation for 5.0×10^-9 mol/L thrombin was 5.0% (n = 7). The plot of the complex formed versus the ratio of thrombin to aptamerⅡindicated 1:1 stoichiometry of the complex. This work demonstrates that the combination of a highly binding aptamer to analyte thrombin based sandwich assay with ECL detection for thrombin analysis is a great promising approach for the determination of protein. Large surface of SWNT provides an amplification platform for carrying a large number of ruthenium complexs, resulting in a sensitive detection of thrombin.