A New Electrochemical Active-inactive Pair Molecular Beacon For Its Application Of Direct Protein Recognition

Proteins are fundamental components of human body, and they are also an important part of life science. They involved in every day activities of human, and are related to nutrition, cells conformation, enzyme, hormone, virus, immunity reaction, substance transformation, heredity, origin of life and evolution. They are viral part of cells. Enzyme, hormone, antibody, transportation molecule, even the framework of cells are composed by proteins. The weight of protein is 40%-50% of biological organs (without water). Proteins are playing an important role in all kind of life function. Therefore, protein analyses is the fundamental subject in life science. Protein quantities analyses is a vital part of protein study, and thus is quite meaningful for human nutriology, disease diagnosis, drug and food analyses. The development of life science is calling for more sensitive, stable and convenient protein analyses methods.Aptamer is a kind of oligonucleotide that had high affinity and selectivity to protein. Comparing with the usual protein analytical methods, aptamer has its unique advantages. Molecular beacons (MB) applied with aptamer sequence becomes an excellent tool for protein analyses. We represent here a novel electrochemical aptamer beacon which could detect bio-molecules directly in solution with its electro-active/inactive pair signal molecules. And a new kind of protein sensor based on this electrochemical aptamer beacon and magnetic nanobeads(MNBs) was designed to human thrombin quantities analyses. This protein sensor can detect a wide range of targets with high selectivity and high sensitivity. With the quick and convenient advantages of electrochemical methods, this peotein sensor may have wide application in biotechnology researchs.The dissertation includes four chapters:Chapter 1 PrefaceThe purpose and development of protein analyses were introduced firstly, and electrochemical methods was focused on. Than aptamer and its advantages in protein analyses were summarized. Molecular beacons and its different detection methods were also included. Magnetic nanobeads and its application in bio-analyses was introduced. At last the purpose and innovation point of the dissertation were pointed out.Chapter 2 Study on the spectra and electrochemistry behavior of Carminic acidAn anthraquinone pigment, carminic acid(CA) was studied by spectra and electrochemical methods. According to the UV-vis absorption spectra of CA, the pKas were calculated. Results showed that in neutral medium, CAs were most of H₃CA^2-/H₂CA^3- conformation, and had the tendency of forming intermolecular H-bond and associate. The electrochemical study showed CAs had sensitive electrical response, and its reduction process went on a deaglycone process. After deaglyconation, two CAs associated and become electro-inactive. At last, we calculated carmine's(CAR) coordinate constant and estimate the association constant of CAs to be lower than 344.Chapter 3 A new electrical active-inactive pair aptamer beacon for its application of direct protein recognition in solutionWe used carminic acid as signal molecules of an aptamer beacon. And proved that it could behave electro-active/inactive due to the conformational shifting of the beacon. Then we combined this electrochemical aptamer beacon with magnetic nanobeads, designed a novel electrochemical protein sensor and applied it into thrombin detection. Magnetic separation and amplification effect enabled our sensor amuch higher sensitivity. The linear range for the determination of thrombin was 7.76×10^-12 mol/L-1.176×10¹⁰ mol/L with a detection limit of 1.29×10^-12 mol/L. also, this sensor could easily be regenerated. Results showed that this novel electrochemical protein sensor has high sensitivity and high selectivity, and its detection methods were simple and quick. It has a potential application in biomedicine and so on.Chapter 4 The thermodynamic study of the novel electrochemical protein sensor and its interaction with thrombinAptamer has high affinity to its target. The study on its interaction with protein reveals its binding mechanism with its target, and is thus helpful in aptamer design, screening and working condition optimization. We analyzed the thermodynamic data of the electrochemical protein sensor and its binding thrombin process. Result showed that the senor binds thrombin spontaneously, this in another way proved the senor's high affinity to thrombin. And the stem-loop structure of the aptamer beacon obstruct the sensor's binding reaction a little, gave the sensor a higher selectivity and lower the background of the detection. This thermodynamic study provided a new method for the study on the interaction between aptamer and its target.