| Electrochemical biosensors are designed to quantitatively produce electrical signals related to analyte concentration and apply electrochemical techniques to biological processes,improving sensitivity and selectivity when detecting ultra-low concentrations of analytes is a core issue in the field of biosensor research.The realization of high sensitivity is to amplify the event of identifying the analyte and enhance the intensity of the output signal,so many signal amplification strategies have been developed.Among them,the signal amplification technology based on the polymerization reaction is the main method for detecting molecular recognition events,such as the polymerase chain reaction(PCR)as a vitro nucleic acid amplification technology can be used to observe and judge the target gene or a DNA fragment with the naked eye.Recently,the polymerization-assisted signal amplification strategy initiated by target has been developed as a novel biosensor mechanism.The use of long-chain polymer materials increases the intensity of the output signal,which in turn directly converts the event of identifying the analyte into a measurable physical signal,thus improves the detection sensitivity and selectivity.The strategy is to couple initiators to biological macromolecules before the recognition event occurs,and in situ polymerization occurs after the recognition event,because the polymer produced is large enough,it can be detected with the naked eye or can directly change the optical and electrochemical properties of the raw materials through the attachment of signal molecules,so that produces signals are easy to distinguish from the background.Common biochemical recognition elements in biosensors include:biotin-avidin,antigen-antibody,DNA double-strands,etc.But they have the disadvantages of small quantity,high cost,short shelf life and variability.In response to this problem,molecularly imprinted polymers(MIPs)synthesized by molecular imprinting technology are used as recognition elements to simulate natural molecular recognition events.MIPs are a new type of polymer material that is stable in nature and can still maintain good recognition ability under harsh conditions such as organic solvents and high temperatures.It can effectively avoid the high cost and environmental sensitivity problems caused by the natural recognition process.Electro-polymerization is a method for preparing MIPs films because of its advantages such as simple preparation,the use of materials of different shapes/sizes,and the ability to control film thickness.In this dissertation,electro-polymerized molecularly imprinted polymers(E-MIPs)are used as an artificial receptor to replace natural biomolecule receptors to generate specific molecular recognition events,in order to improve the selectivity and stability of the sensor and reduce the manufacturing cost.In this dissertation,the electrochemical biomimetic sensors of theophylline and sialic acid imprinting were respectively prepared by combining the above two strategies of molecularly imprinted polymers film and polymerization-assisted signal amplification.The surface-initiated atom transfer radical polymerization(SI-ATRP)is a reversible deactivation/“living”technique in which the growth of surface grafted polymers can be directly monitored by atomic force microscopy(AFM).Compared with the sensor based on natural recognition event-triggered polymerization-assisted signal amplification,this method not only avoids its disadvantages to some extent,but also has the advantages of low detection limit,wide detection range,low cost,and good stability.The details are as follows:1.Preparation of theophylline molecular imprint sensor based on polymerization reaction assisted signal amplification technologyUsing thiophene-3-acetic acid as the functional monomers,theophylline as the templates and the electro-polymerized molecularly imprinted polymers film were used as the artificial receptor of theophylline and its preparation conditions were optimized.The results show that the imprinting effect of the obtained polymer films is remarkable.Through the specific recognition of the artificial receptor towards the initiator-theophylline complex,it was immobilized on the electrode surface and triggerring SI-ATRP.Acrylamide(AM)was used as the chain growth unit,and the situ growth of polyacrylamide(PAM)monitored by AFM technology,the results showed that the polymer chain obtained was controllable within a certain period of time.PAM provides a large number of binding sites for the electrochemical tag sodium phenothiazine sulfonate(PTZ-343),thus enhanced sensitivity.The developed sensor displays a lower detection limit(1.1×10-11 M),a wider detection range(0.3×10-10-0.3×10-4 M),excellent selectivity,reproducibility and stability.2.Preparation of sialic acid molecular imprint sensor based on polymerization reaction assisted signal amplification technologyWith O-phenylenediamine as functional monomer,sialic acid as template,electro-polymerized molecularly imprinted polymers(E-MIPs)film as an artificial receptor,and the imprinting factor was used as a measure to optimize the imprinting conditions,the applicable properties of its toward SA was evaluated by the binding capacity,the results show that the artificial receptor has excellent rebinding performance.After the rebinding reaction,the SI-ATRP initiator reacts with sialic acid to immobilize it in situ,using glycidyl methacrylate(GMA)as the chain growth units and initiating the polymerization reaction.The growth of polyglycidyl methacrylate(PGMA)leads to a large number of couplings of the electrochemical tag aminoferrocene(Fc NH2),the synthesis and growth of the polymer were characterized by X-ray photoelectron spectroscopy(XPS)and AFM techniques,and the results indicate that PGMA was successfully synthesized and controllability for growth.The proposed electrochemical biomimetic sensor exhibits excellent performance of electrochemical analysis with a linear range of 1×10-8-1×10-3 M and a detection limit of5.2×10-9 M. |
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