Thrombin And Cocaine Electrochemical Aptamer-Based Biosensors

Determination of protein has been a topic of significant interest with the accomplishment of Human Genome Project and the rapid progress of proteomic strategies.Intense research activities are carried out to develop rapid,simple,specific and sensitive detection devices for protein in medical diagnostics and biomedical research application,while the detection methodologies for protein based on antibodies cannot meet the demand.Aptamers have been emerging as a new protein recognition element in wide range of bioassays.Aptamers have attracted a considerable attention due to their ability to bind target protein with high affinity and specificity and have many advantages over antibodies.These properties of aptamers make aptamers ideal candidates as protein recognition elements in a wide range of bioassays and for the development of diseases diagnostics. Electrochemical aptamer-based biosensors have attracted significant interest due to its simple, sensitive and low-cost detection capability for applications including clinical genetic analysis, environment monitoring,and forensic identification.Nanoparticles are of considerable interest owing to their unique physical and chemical properties, and offer excellent prospects for biosensor.The power and scope of nanoparticles can be greatly enhanced by coupling them with the high specificity of the aptamer and the high sensitivity of the electrochemical recognitions.The aim of the present work is to develop novel electrochemical biosensors based on nanoparticles and aptamer.Labeled and label free electrochemical aptamer-based biosensors were designed for the determination of biological molecules such as protein and drug with high sensitivity and selectivity.This thesis consists of two parts.First part,chapter 1,is general introduction while second part, consisting of four chapters,is research report.Chapter 1-General introduction.In this chapter,the concepts of aptamer,including in vitro selection of aptamer,the characteristic of aptamer,the interactions between aptamer and target, chemical modification of aptamers,were described;the principle of aptamer-based biosensor and electrochemical aptamer-based biosensor were presented;the recent developments of labeled and label-free electrochemical aptamer-based biosensor were reviewed;the purpose and the significance of this research work were simply presented.Chapter 2-Labeled electrochemical aptamer-based biosensor for the determination of thrombin. A highly sensitive electrochemical method for the detection of thrombin based on the two thrombin-binding-aptamers(TBAâ… and TBAâ…¢) has been developed.TBAâ… with a thiol group at the 3′-terminal by self-assembled onto the surface of a gold electrode was employed as an electrochemical probe for capturing thrombin.TBAâ…¢labeled with carbon-nanotubes(CNTs) tagged with silver nanoparticles(CNTs-Ag) was used for the detection of thrombin.CNTs were decorated with a large number of silver nanoparticles to form CNTs-Ag.Binding events were monitored by differential pulse voltammetric(DPV) signal of silver nanoparticles.There was a linear relation between the peak current at+0.20 V(vs.Ag/AgCl) and thrombin concentration over the range from 0.012 to 10 nmol/L with a detection limit of 0.005 nmol/L of thrombin.The detection processes are simple due to no requirement of acid-dissolving the label metal,the presence of other protein,as BSA did not affect the detection.This work demonstrated that the probe labeled with CNTs loaded with silver nanoparticles offers a great promising approach for sensitive detection of protein.Chapter 3-Label free electrochemical aptamer-based biosensor for the determination of thrombin. A simple and highly sensitive electrochemical impedance spectroscopy(EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin.The signal enhancement was achieved by using gold nanoparticles(GNPs),which was electrodeposited onto a glassy carbon electrode(GCE),as a platform for the immobilization of the thiolated aptamer.In the measurement of thrombin,the change in interfacial electron transfer resistance of the biosensor using a redox couple of[Fe(CN)₆]^3-/4- as the probe was monitored.The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12 nmol/L to 30 nmol/L.The association and dissociation rate constants of the immobilized aptamer-thrombin complex were 6.7×10³ M^-1 s^-1 and 1.0×10^-4 s^-1, respectively.The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed.This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein.This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.The obtained kinetics parameter and equilibrium constants will direct us to find out the basic principle for designing the aptamer based biosensor and to deeply understand the properties of the aptamer toward protein,and it will be great importance of developing new approaches for disease diagnosis.Chapter 4-Label free electrochemical aptasensor for the determination of cocaine.A simple and highly sensitive impedance spectroscopy biosensor based on aptamer as molecular recognition element was developed for the determination of cocaine.The solution of the modified aptamer for cocaine and the complementary short DNA were first mixed in the buffer in equal more quantities to treat a half-duplex,and then the half-duplex aptamers were immobilized onto a gold electrode surface via thiol-Au interactions.The electroactive complex,[Ru(NH₃)₆]³⁺,which can bind to anionic phosphates of aptamer and DNA strands completely through electrostatic interactions,serves as a signaling probe.[Ru(NH₃)₆]³⁺ is adsorbed on a half-duplex aptamers backbone,giving a clear detection signal in admittance.After binding with cocaine,the complementary short DNA were released together with the[Ru(NH₃)₆]³⁺ that was electrostatically binding to them,resulting in a lower admittance signal for the[Ru(NH₃)₆]³⁺ confined on the electrode surface.The admittance response exhibits a linear relationship to the logarithm of the target concentration in the concentration range from 2μmol/L to 100μmol/L.This device combines the selectivity of the aptmer recognition layer with the sensitivity of admittance to the determination of cocaine.Chapter 5-Labeled electrochemical aptamer-based biosensor for the determination of cocaine.A novel electrochemical self-assembled onto the surface of a gold electrode incorporating a signal enhancement for the determination of cocaine was designed.Gold nanoparticles were self-assembled onto the surface of a gold electrode through 1,6-hexanedithiol.A bifunctional derivative of the 32-base cocaine-binding aptamer with a redox-active ferrocene moiety and a thiol linker group at the termini of the strand was self-assembled onto the surface of gold nanoparticles.The oxidation peak current is linearly related to the concentration of cocaine from 1.0 to 15.0μmol/L with a detection limit of 0.5μmol/L.It was found that the sensitivity of the aptamer-based biosensor with gold nanoparticles modification is～10-folds higher than that of the aptamer-based biosensor without gold nanoparticles modification.The apparent heterogeneous electron transfer rate of ferrocene was determined to be 67 s^-1.Compared with the apparent heterogeneous electron transfer rate of ferrocene at bare gold electrode,the apparent heterogeneous electron transfer rate of ferrocene increased at gold nanoparticles modified gold electrode.This work demonstrates that gold nanoparticles-assembled gold electrode provides a promising platform for immobilizing aptamer and enhancing the sensitivity.