Studies On Electrochemical Aptasensor For Thrombin Dectrion

Thrombin, a kind of serine protease that involved in thrombosis and hemostasis, plays an important role in revealing tumorigenesis mechanism and judgment of early diagnosis, curative effect and prognosis. Since the concentration of thrombin in blood reaches to nmol·L-1, create a simple, rapid and high-sensitivity method for detection of thrombin is extremely important. Electrochemical aptasensor, the combination of SELEX-screened aptamers and electrochemical sensors, integrates the advantages of high sensitivity, fast response, simple operation and low cost of electrochemical sensors as well as high selectivity and specificity of aptamer, which thus provids very broad application prospects in the field of thrombin detection. The aim of present work is to develop simple, reliable, fast, high-sensitivity electrochemical aptasensor for the detection of thrombin. Therefore, this work focuses on the construction of the sensitive interface, the preparation of multi-functionalized nanoparticles and the development of novel electrochemical signal amplificatory strategy. The detail contents are as follows:In chapter one, after general introduction of aptamer including its in vitro selection, characteristic and interaction with thrombin, the principles, classification, immobilization method and associated electrochemical analysis techniques of the electrochemical aptasensor were described in detail. Moreover, the application of signal amplificatory strategy for electrochemical aptasenor was highlighted. Lastly, the aim and the significance of this thesis were briefly introduced.In chapter two, since the aptamers are intrinsically unable to act as redox partners in an electrochemical reaction, most electrochemical aptasensors are relied on the label of either aptamer or its complementary strand. The labeling process, however, is complicated, time-consuming, and may affect the biological activity of aptamer. In this experiment, we described a signal-off and label-free electrochemical aptasensor for the detection of thrombin based on{nano-Au/Thi}n multilayer films acted as redox probe and thrombin aptamer (TBA) immobilized matrix. Herein, the {nano-Au/Thi}n multilayer films are formed on the electrode surface via the layer-by-layer self-assembly technique, which avoids the fuzzy labeling process of aptamer, thus leads to a great increase of redox probe amount on electrode surface and a decrease of distance between the redox probe and electrode surface to a certain extent. This approach possesses some dramatic advantages, such as simple operation, fast response, high specificity and so on. At the same time, the constructing method of this aptasensor is applicable to many other aptamers, providing very broad application prospects in the field of protein detection and disease diagnosis.In chapter three, hemin/G-quadruplex used for enzyme catalysis can effectively amplify the electrochemical signal. Compared with protein enzymes, hemin/G-quadruplex is relatively easy to label, less expensive to produce, and more stable against hydrolysis and heat treatment. Take advantage of the G base-riched thrombin aptamer can combine with hemin to form a horseradish peroxidase-like hemin/G-quadruplex catalytic structure, we described a signal-off electrochemical aptasensor with electrocatalytic amplification for the detection of thrombin. Moreover, the new strategy of employing HRP to block the possible remaining active sites and thus then amplify the response of the TBA-thrombin recognition was proposed. The proposed electrochemical aptasensor realized the recognition to thrombin with high specificity and high sensitivity, and avoided the fuzzy labeling process of electroactive substance and the enzyme. Experimental studies have shown that this approach is simple, practical and greatly improved the sensitivity of electrochemical aptasensor.In chapter four, compared with protein enzyme, the nano-metals for electrocatalytic amplification possess better thermal stability. In this experiment, we described a target-induced signal-on electrochemical aptasensor for the detection of thrombin based on the dual-amplification of inert material stearic acid OCA and dual-functional Au-PtNPs. With the addition of H2O2to the electrolytic cell, the Au-PtNPs on the aptasensor surface effectively electrocatalyzed the oxidation-reduction reaction of methylene blue MB, which provided an extremely amplified electrochemical signal. When the quantitative analysis was carried out, the OCA label TBA on electrode surface captured thrombin and followed with the leave of the electrode surface, which led to a decrease in steric hindrance of electrode surface and an increase in catalytic efficiency of Au-PtNPs, significantly amplified the electrochemical signal, and thereby effectively improved the sensitivity of electrochemical aptasensor. The proposed aptasensor has been demonstrated to offer a simple, rapid and sensitive method for the detection of thrombin, providing a satisfactory result in clinical detection.In chapter five, a novel3,4,9,10-perylenetetracarboxylic acid/hemin nanocomposites (PTCA/hemin) with a pair of well-defined redox peaks and intrinsic horseradish peroxidase-like activity was prepared through π-π interactions. And a pseudobienzyme-channeling amplified signal-on electrochemical aptasensor based on glucose oxidase (GOx) as blocking reagent and PTCA/hemin nanocomposites simultaneously as redox probes and electrocatalysts was prepared for the detection of thrombin. The superior membrane-forming property of PTCA/hemin nanocomposites not only avoids the conventional fussy process for redox probe immobilization, but also reduces the participation of the membrane materials that act as a barrier of electron transfer, providing a simple and sensitive method of detection of thrombin. Moreover, the new strategy of employing GOx to block the possible remaining active sites and thus then construct pseudobienzyme-channeling amplified system for amplifying the response of the TBA-thrombin recognition was proposed, which further improved the sensitivity of electrochemical aptasensor.In chapter six, since bienzyme catalytic signal amplification is obviously larger that of single enzyme catalytic system, we described a pseudobienzyme amplifying sandwich-type electrochemical aptasensor for the detection of thrombin with hemin/G-quadruplex simultaneously serve as NADH oxidase and an HRP-mimicking DNAzyme. Compared with protein enzymes labeled aptasensor, the proposed aptasensor are relatively easy to label, less expensive to produce, and more stable against hydrolysis and heat treatment. More importantly, this strategy realized the simultaneous action of the hemin/G-quadruplex as an NADH oxidase and HRP-mimicking DNAzyme in bioelectrocatalytic amplification, which avoided the fussy labeling process, resolved the spatial distribution of each sequentially acting enzyme and provided a new strategy for constructing simple and sensitive aptasensor for the detection of thrombin.