Studies On Electrochemical MicroRNA Biosensors Based On Nanotechnique And Biotechnique For Signal Amplification

Electrochemical microRNA biosensors are novel and valuable tools which combines complementary base pairing principle and electrochemical analysis. It holds advantages of high sensitivity, good selectivity, rapid response and simple operation. MicroRNAs are typical human cancer biomarkers. In order to achieve early diagnosis of cancer, the ultrasensitive detection of microRNA is essential. In recent years, a variety of amplification technology, such as nanomaterials, biological and chemical combination, are widely used to fabricate microRNA biosensor. Therefore, this research focuses on the construction of sensitive interface, the application of enzyme catalysis, the preparation of functionalized nanomaterials, the realization of target-catalyzed recycling and the application of novel signal amplification strategies to construct electrochemical microRNA biosensor. The research contents are mainly as follows:1. A novel label-free electrochemical microRNA biosensor using Pd nanoparticles as enhancer and linkerIn this work, a novel biosensor for microRNA-155 detection based on the conductive self-assembled multilayer comprised of nafion, thionine (thi) and Pd nanoparticles (PdNPs) was successfully prepared. The introduction of PdNPs focuses on two aspects:one is as linker to combine capture probe, the other is as enhancer to amplify electrochemical signal. Nafion was firstly dropped to a bare glass carbon electrode. Then thi was absorbed by the cation exchanger nafion. Furthermore, a PdNPs layer which was used to immobilize target biomolecules was constructed by combining with the amino group of thi. The proposed biosensor showed excellent electrocatalytic activity towards H2O2 and enhanced the current response of the biosensor. The characterization of nanomaterials (PdNPs) and the performance of the microRNA biosensor were studied. The resulting biosensor presented high sensitivity, good stability and a broad linear reponse from 5.6 to 5.6×105 pmol·L-1 with the detection limit of 1.87 pmol·L-1 under optimization of the assay condition.2. Dual signal amplification strategy for enzyme-free electrochemical detection of microRNAsIn this work, the combination of catalyzed hairpin assembly reaction (CHA) and hybridization chain reaction (HCR) is introduced to develop an electrochemical biosensor without the assistance of enzyme for highly sensitive microRNA detection. Target recycling was realized by CHA, which broke the limitation of 1:1 binding of target microRNA and capture probe and led to signal amplification. It is worth mentioning that target recycling in this work is achieved by strand displacement instead of any nuclease, which can simplify the experimental operation and avoid the deactivation of enzyme. DNA propagation was completed by HCR. A large amount of electron transfer mediators intercalated into the minor groove of the propagated dsDNA polymers to achieve enhanced electrochemical signal. Moreover, gold nanoparticles-graphene nanohybrid (Au-Gra) with excellent conductivity, good biocompatibility and high surface area was modified on the surface of glassy carbon as the sensor platform to increase the immobilization of capture probes. Owing to the good platform and dual signal amplification strategies, detection sensitivity of the proposed biosensor enhanced largely, the detection limit is 3.3 finol·L3. An electrochemical biosensor for sensitive detection of microRNA-155:combining target recycling with cascade catalysis for signal amplificationIn this work, a novel electrochemical biosensor based on catalyzed hairpin assembly (CHA) target recycling and cascade electrocatalysis (cytochrome c (Cyt c) and alcohol oxidase (AOx)) for signal amplification was developed for highly sensitive detection of microRNA. Cascade electrocatalysis is based on two specific enzymes. The catalytic production of first enzyme is just the catalytic substrate of second enzyme. We have synthesized porous TiO2 nanosphere, which can offer more surface area for Pt nanoparticles (PtNPs) enwrapping and enhance the amount of immobilized DNA probe 1 and Cyt c accordingly. AOx, labeled on a probe, can be immobilized onto the electrode surface by hybridization. When the ethanol added into the detection buffer, the cascade catalysis amplification was carried out by AOx catalyzing ethanol to acetaldehyde with the concomitant formation of high concentration of H2O2, which was further electrocatalyzed by PtNPs and Cyt c.In addition, target recycling was accomplished by CHA reaction without the assistance of any nucleases, which can effectively simplify experimental operation. The experimental results indicated that the sensitivity of the microRNA biosensor enhanced obviously by the combination of nanotechnique and biotechnique. The detection limit is 0.35 finol·L-1.