Novel Biosensing Methods Based On DNA Nanomachine And Nanomaterials

More and more small molecules,nucleic acids and proteins need to be detected and analyzed by biosensor technology,an important research tool in the field of life science.Biosensor technology has been widely used in medical diagnosis,gene analysis,clinical diagnosis and treatment etc.However,with the growth and progress of society,the old biosensor technology could not fully meet the needs of all fields.It is also a great challenge for researchers to develop new biosensors with strong specificity,high accuracy,short response time and simple operation procedure to meet the practical application in the fields of biomedical and others.With the emergence of nanotechnology and the continuous development of biosensor technology,nanoscience provides a new direction for the development of biosensors.In order to solve the problems existing in biosensors,a series of biosensors with high sensitivity and specificity were constructed based on DNA nanotechnology and nanomaterials for the detection of small molecules.The main contents of the study are as follows:（1）In chapter 2,pH plays a crucial parameter associated with various cellular behaviors and biological processes,and abnormal pH is a typical feature of many diseases.Based on this,we constructed a reversible fluorescent DNA triangular prism nanomachine（TPN）to monitor and map the changes of pH in living cells as the i-Motif sequences of TPN could respond to it.Under neutral or weakly alkaline pH conditions,the i-Motif sequences adopted a random coil with the labeled two fluorophores Cy3 and Cy5 were in spatial separation,resulting that the fluorescence was in an“off”state.Under the condition of acidic pH,the i-Motif sequences in TPN were protonated and folded to form an intramolecular i-tetraplex,which closed proximity between the fluorophores Cy3 and Cy5,resulting in a significant fluorescence resonance energy transfer（FRET）signal with the fluorescence was in an“open”state.This sensing system could not only visualize pH fluctuations under varying stimuli in living cells,but also monitor spatiotemporal pH changes during endocytosis.This TPN possessed wide range of pH detection in the range of 5.0 to 7.5,and had fast response speed,pretty reversibility.（2）The accurate determination and quantification of hydrogen peroxide（H₂O₂）and glucose are necessary for diagnosis and bioengineering.We designed and synthetised g-C₃N₄-MnO₂ nanocomposite based on the graphitic-C₃N₄（g-C₃N₄）and manganese dioxide（MnO₂）nanosheets for the rapid and selective detection of H₂O₂and glucose.In this method,g-C₃N₄ nanosheets had blue fluorescence which could be quenched by MnO₂ due to the FRET between them.The MnO₂ was reduced to Mn²⁺when H₂O₂ was introduced,resulting in an elimination of FRET with the recovery of the quenched fluorescence.This detection system was also suitable for the detection of glucose as H₂O₂ is one of the main products in the oxidation reaction of glucose catalyzed by glucose oxidase.Additionally,the fluorescence was linear with the concentration of H₂O₂ in the range of 0 to 130μM,and also showed a good linear relationship for glucose in the range of 0 to 150μM.The detection limits were as low as 1.5μM.In addition,the proposed sensor showed advantageous of fast detection speed,strong operability,and high identification degree.（3）In chapter 4,organophosphorus（OPs）,an important component of pesticides,the unstandard and unreasonable used of it resulting in the pesticide residues of agricultural products and the environment severely exceeded which will cause great harm to human health and environment.In response to this problem,the g-C₃N₄-MnO₂nanocomposite was synthesized for the detection of OPs.The fluorescence of g-C₃N₄can be quenched by MnO₂ namosheets.In the presence of thiocholine（TCh）,a catalytic hydrolysis product of acetylthiocholine（ATCh）by acetylcholinesterase（AChE）,MnO₂ could be reduced to Mn²⁺with the quenching fluorescence of g-C₃N₄was restored.Interestingly,the produce of TCh would decreased when the OPs existed,as the activity of AChE was greatly inhibited because of the phosphorylation between them.And the MnO₂ was still deposited on g-C₃N₄ nanosheets with the FRET system was keeped.Therefore,we can detect OPs according to the change of fluorescence signal of g-C₃N₄.The synthesis method was simple,and the system could accurately detect the target.