Study On Novel Homogeneous Electrochemical Biosensing Strategies Based On Functionalized Nanomaterials

Recently,food safety issues and disease-related biomacromolecule detection have attracted extensive public attention.Therefore,it is urgent to develop a novel biosensor with high sensitivity and selectivity for such targets detection.However,traditional electrochemical biosensors need the modification on the electrode surface.The recognition between capture probes and targets suffers from low binding efficiencies originating from spatial hindrance of the interface.In order to solve these problems,two novel homogeneous electrochemical biosensors in this dissertation have been developed based on functionalized nanomaterials for the detection of food toxin and biomacromolecule.Due to the advantages of nanomaterials and DNA aptamer,the developed biosensors have been demonstrated high sensitivity and selectivity for targets dection.The main contents are as follows:?1?A novel gap-electrical homogeneous aptasensor based on magnetic beads coated with Au nanoparticles?Au@Fe₃O₄?as conductive bridges to mediate signal transduction has been proposed for monitoring ochratoxin A?OTA?.In this strategy,the probe DNA modified on the surface of Au@Fe₃O₄ has been hybridized with the OTA aptamer to form a duplex DNA.When the target OTA exists,the strong binding force between the aptamer and OTA will induce the aptamer to dissociate from the aptamer-DNA duplex to form an OTA-aptamer complex.Exonuclease??Exo??in the reaction solution will gradually digest the aptamer in the OTA-aptamer complex into mononucleotide,and release OTA to trigger more reactions to achieve the signal amplification.At the same time,Exo?will also digest the single-strand capture probe DNA on the surface of Au@Fe₃O₄,making the activity of terminal transferase?TdT?ineffective.Under magnetic separation,Au@Fe₃O₄ is transported on the insulated gap of electrodes to form conductive bridges,which improve the conductivity of microelectrodes.However,in the absence of OTA,the duplex DNA on the surface of Au@Fe₃O₄ cannot be digested by Exo?.Upon the addition of TdT transferase,such double DNA will be prolonged at the 3'end of the capture probe.Due to amounts of DNA wrapped Au@Fe₃O₄,the collected Au@Fe₃O₄ after magnetic separation exhibited poor conductivity on the surface of the microelectrodes.Under the optimal experimental condition,the developed aptasensor exhibited high selectivity and sensitivity for OTA assay in the response range from1 ng/mL to 1?g/mL with a correlation coefficient of 0.988 and the detection lower limit reaches 0.85 ng/mL.?2?A novel label-free electrochemical biosensor technology based on black phosphorus-induced aggregation of copper ion mediated signal transduction has been proposed for phosphatase activity assay.Black phosphorus is a two-dimensional semiconductor structure,which has been demonstrated the specifically adsorbing copper ions.In this strategy,the presence of pyrophosphatase can hydrolyze of the substrate pyrophosphate,thus inhibiting the formation of pyrophosphate-copper ion complex.Copper ions in the reaction solution can be effectively adsorbed on the surface of black phosphorus-modified glassy carbon electrode?BP-GCE?,and a strong copper ion redox peak is detected.On the contrary,when pyrophosphatase does not exist,the strong interaction between pyrophosphoric acid and copper ions prevents copper ions in the reaction solution being adsorbed on the surface of BP-GCE,thus almost no electrochemical signals of copper ion is detected.The experimental results show that the electrochemical signal of copper ion is proportional to the logarithm concentration of pyrophosphatase,and the biosensor can monitor the inhibitory effect of external factors on the activity of pyrophosphatase.Based on the above method,the optimal experimental results show that the sensitive detection of pyrophosphatase can be achieved,the response range is from 0.01 mU/mL to 10 mU/mL with a correlation coefficient of 0.986 and the detection limit is 0.009 mU/mL.