Studies On Ultrasensitive Electrochemical Biosensors Of Adenosine And MicroRNAs Based On Signal Amplification Technology

Electrochemical biosensor is a new detection device that employs biological molecules as recognition element and combines with electrochemical detection technology. In recent years, electrochemical biosensor has attract substantial research efforts in diseases diagnostics, drug analysis, environmental monitoring and food analysis, due to the remarkable features of simple equipment, high sensitivity, good selectivity and fast response. However, the concentration of the analyte is usually very low in actual testing, which requires urgently researchers to develop various signal amplification strategies to improve the sensitivity of sensor. Therefore, aiming to simplify the operation steps and design different signal amplification methods, a series of highly sensitive electrochemical biosensors for adenosine and MicroRNAs in this thesis. Furthermore, the corresponding principle and performance of each work have been explored carefully. The main research work are summarized as follows: Part 1 Target-induced strand release and thionine-decorated goldnanoparticle amplification labels for sensitive electrochemical aptamer-based sensing of small molecules.A sensitive and selective electrochemical aptamer sensing platform for adenosine based on target-induced strand release and gold nanoparticle(AuNP) amplification labels is developed. The association of the target adenosine with the corresponding aptamer leads to the release of the thiol c-DNA strands from the aptamer/c-DNA duplexes. The released thiol c-DNA strands are captured on a gold sensing electrode by their complementary sequences through DNA hybridizations. The AuNPs are then attached to the surface-hybridized thiol c-DNA strands via Au-S bond. These AuNPs further adsorb a large number of electroactive species, thionine, on their surfaces, which leads to significantly amplified current responses for highly sensitive monitoring of adenosine down to 0.05 nM. Besides, the proposed method shows excellent selectivity toward adenosine against other analogous interference molecules due to the high specificity of the aptamer recognition capability. Moreover, by changing the corresponding specific aptamer recognition strands, the proposed method can offer a more general sensing platform for the detection of low levels of various types of biomolecules(proteins, cells, amino acids, etc.). Part 2 In Situ DNA-Templated Synthesis of Silver Nanoclusters for Ultrasensitive and Label-Free Electrochemical Detection of MicroRNA.On the basis of the use of silver nanoclusters(AgNCs) in situ synthesized by cytosine(C)-rich loop DNA templates as signal amplification labels, the development of a labelfree and highly sensitive method for electrochemical detection of microRNA(miRNA-199a) is described. The target miRNA-199 a hybridizes with the partial dsDNA probes to initiate the targetassisted polymerization nicking reaction(TAPNR) amplification to produce massive intermediate sequences, which can be captured on the sensing electrode by the self-assembled DNA secondary probes. These surface-captured intermediate sequences further trigger the hybridization chain reaction(HCR) amplification to form dsDNA polymers with numerous C-rich loop DNA templates on the electrode surface. DNA-templated synthesis of AgNCs can be realized by subsequent incubation of the dsDNA polymer-modified electrode with AgNO3 and sodium borohydride. With this integrated TAPNR and HCR dual amplification strategy, the amount of in situ synthesized AgNCs is dramatically enhanced, leading to substantially amplified current response for highly sensitive detection of miRNA-199 a down to 0.64 fM. In addition, the developed method also shows high selectivity toward the target miRNA-199 a. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective, and simple detection of different types of microRNA targets. Part 2 Multiplexed and amplified electronic sensor for the detection of microRNAs from cancer cells.The detection of microRNA expression profiles plays an important role in early diagnosis of different cancers. Based on the employment of redox labels with distinct potential positions and duplex specific nuclease(DSN)-assisted target recycling signal amplifications, we have developed a multiplexed and convenient electronic sensor for highly sensitive detection of miRNA-141 and miRNA-21. The sensor is constructed by self-assembly of thiol-modified, redox species-labeled hairpin probes on the gold sensing electrode. The hybridizations between the target miRNAs and the surface-immobilized probes lead to the formation of RNA/DNA duplexes, and DSN subsequently cleaves the redox-labeled hairpin probes of the RNA/DNA duplexes to recycle the target miRNAs and to generate significantly amplified current suppression at different potentials for multiplexed detection of miRNA-141 and miRNA-21 down to 4.2 fM and 3.0 fM, respectively. The sensor is also highly selective toward the target miRNAs and can be employed to monitor miRNAs from human prostate carcinoma(22Rv1) and breast cancer(MCF-7) cell lysates simultaneously. The sensor reported here thus holds great potential for the development of multiplexed, sensitive, selective and simple sensing platforms for simultaneous detection of a variety of miRNA biomarkers for useful clinic applications with careful selection of the redox labels.