The Application Of Functional Nanomaterials And Amplification Strategies In Electrochemical Aptasensor

The technology of electrochemical sensor is the most wide application and the most perfect method of sensor analysis, which possesses some conspicuous advantages, such as, low cost for detection, fast analysis speed, high sensitivity, simple operation, easy automation and real-time monitor, ect.on. Electrochemical aptasensor is a kind of electrochemical biosensor that based on aptamer as molecular ident ification element. The detection principle is that after aptamer combining with the target, the electrochemical signal would change, which could be as the basis of detection. Therefore, electrochemical aptasensor has obtained increasing attention due the fact that it units the advantages of aptamer and electrochemical analysis. And the new generation of electrochemical aptasensor that incorporated aptamer identif ication element and electrochemical converter further riched the traditionally electrochemical detection method for biomolecule gradually. In recent year, to improve the sensitivity of electrochemical aptasensor, the researches that focus on novel mutilfunctional nanomaterials, various new amplification strategies and catalysis have raised widespread concerns, which is very important for the construction of aptasensors with high efficient, ultrasensitive, high selectivity, reliable and practical to realize the detection of target in clinic. Hence, a series of electrochemical aptasensor were constructedbased on the preparation of the novel mutilfunctional mater ials, and the innovation of signal amplif ication technology and catalys is. The details are mainly as follows: 1. Cleavage-Based Hhybridization Chain Reaction for Electrochemical Detection of ThrombinIn the present work, we constructed a new label-free “inter-sandwich” electrochemical aptasensor for thrombin（TB） detection by employing a cleavage-based hybridization chain reaction（HCR）. An novel single-stranded DNA（defined as binding DNA） was designed, which contained the thrombin aptamer binding sequence, a DNAzyme cleavage site and a signal reporter sequence. In the absence of a target TB, the designed DNAzymes could combine with the thrombin aptamer binding sequence via complementary base pairing, and then Cu2+ could cleave the binding DNA. In the presence of a target TB, TB could combine with the thrombin aptamer binding sequence to predominantly form an aptamer-protein complex, which blocked the DNAzyme cleavage site and prevented the binding DNA from being cleaved by Cu2+-dependent DNAzyme that owns the advantages of not easy to lose activity, high effient for cleavage. Therefore the cost and operation procedure for this experiment decreased. As a result, the bindingDNA could leave the electrode surface to trigger HCR with the help of two auxiliary DNA single-strands, A1 and A2. Then, the electron mediator hexaammineruthenium（III） chloride（[Ru（NH3）6]3+） was embedded into the double-stranded DNA（ds DNA） to produce a strong electrochemical signal for the quantitative measurement of TB. The introduction of HCR not only immobilized electroactives on the electrode, but also increases the immobilization amount, which could amplify the electrochemical signal. For further amplification of the electrochemical signal, grapheme reduced by dopamine（PDA-r GO） was introduced as a platform in this work. Meanwhile, the adopted of this nanomaterials could improve the sensitivity of this aptasensor. With this strategy, the aptasensor displayed a wide linearity in the range of 0.0001 nmol·L-1 to 50 nmol·L-1 with a low detection limit of 0.05 pmol·L-1. Moreover, the resulting aptasensor exhibited good specificity and acceptable reproducibility and stability. Because of these factors, the fabrication protocol proposed in this work may be extended to clinical application.2. A Novel Nanomaterials 3,4,9,10-Pe rylenetetracarboxylic acid/O-phe nylenediamine as Redox Probe for Electrochemical Aptasensor System Based on Fe₃O₄ Magnetic BeadIn this work, a novel redox probe 3,4,9,10-perylenetetracarboxylic acid/O-phenylenediamine（PTCA/OPD） with large surface area, good membrane-forming and stability was successfully prepared, and applied to design a nonenzymatic catalyst signal amplification strategy based Fe₃O₄ magnetic bead（MB） for electrochemical aptasensor to detect thrombin（TB）. Importantly, the theoretical calculation was used to demonstrate that the synergistic action causing by π-π interactions and hydrogen bonding between PTCA and OPD increased the delocalize charge and resonance energy effectively, finally making the miscellaneous peaks of PTCA tended into a pair of well-def ined redox peaks acting as redox probe. In general, the present work not only highlighted a novel redox probe with highly electrochemical activity and conductivity, but also endowed MB with new application in artificial enzyme mimetics and electrocatalysis. The prepared aptasensor exhibited a wide linear dynamic range from 0.0001 to 30 nmol·L-1 toward TB with a low detection limit of 0.05 pmol·L-1. Impressively, the investigation between PTCA and OPD will be important basis in pursuit of multifunctional nanomater ials for designing new and efficient electrochemical sensing and related systems. 3. Synthesis of Novel Multifunctionalized Peryleneteracarboxylic Supramolecule and Its Application in AptasensingIn this work, a series of novel multifunctionalized peryleneteracarboxylic supramolecules were synthesized based on hydrogen bonding interaction between 3,4,9,10-perylenetetracarboxylic acid（PTCA） and amines, which possess large specific surface area, good membrane-forming property and high stability. Importantly, an interesting phenomenon was found that these series of supramolecules could reconcile self-derived farraginous redox peaks of PTCA and obtain a pair of well-def ined redox peaks, which were able to act as redox carrier for char ge-generating and electron-transporting. And the probable mechanisms for this phenomenon was firstly discussed through integrating theoretical with practice researches. To further reveal the advantages of these novel multifunctionalized supramolecule nanomater ials, PTCA/triethylamine（PTCA/TEA） was chosen as the best candidate of redox carrier to participate in a “signal-on” aptasensor for thrombin（TB） detection by employing Fe₃O₄ magnetic beads（MB） as well mimic enzyme to catalyze the PTCA/TEA for signal amplif ication. As a result, a wide linear detection range of 0.0001 nmol·L-1 to 50 nmol·L-1 is acquired with a relatively low detection limit of 0.05 pmol·L-1. And the proposed aptasensor exhibited good specificity and acceptable reproducibility and stability. After all, the explorations between PTCA and amines would set up a meaningful basis in seeking multifunctionalized supramolecule nanomaterials based on PTCA for expanding the application of PTCA in more wide fields.