Novel Electrochemical Biosensing Research Based On Modified Glass Nanopore

Electrochemical biosensors are widely used in the field of biosensors,which can convert biochemical signals.Its working principle is based on the recording of the electrical signal generated when the bioreceptor selectively reacts with the target analyte,and the electrical signal is closely related to the conformation and concentration of the analyte.As a kind of electrochemical biosensor,glass nanopore sensor has developed rapidly in the field of sensing due to its advantages of easy preparation,easy operation,and miniaturization.Resistive-pulse sensing and ionic current rectification in electrochemical detection techniques are two commonly used signal detection methods for nanopore.Nowadays,the combination of glass nanopore and electrochemical detection techniques has been used by more and more scholars to observe nano-scale chemical and biological phenomena,which has promoted extraordinary progress in science and medicine.In this paper,a glass nanopore and electrochemical detection techniques are combined to construct a glass nanopore sensor for detecting small molecule and studying the signal of DNA translocation,which realizes the label-free detection of cyclic adenosine monophosphate（cAMP）and the signal study of long-chain DNA translocation in different conformations.The specific research content is as follows:（1）Glass nanopore sensor for label-free detection of cyclic adenosine monophosphate.As a second messenger,cAMP is an important molecule used for signal transduction in cells.It responds to certain extracellular signal molecules and is released by the cell to trigger a series of specific cellular physiological changes.Therefore,exploring the changes in cAMP can provide valuable information for understanding the signal transduction that occurs inside the cell.In Chapter 2,we designed a glass nanopore sensor with a cAMP-specific antibody modified on the nanopore wall,and combined with the ionic current rectification to achieve specific,sensitive and label-free detection of the second messenger cAMP.We first used a laser-based puller to pull glass capillary to prepare glass nanopore（with a diameter of about60 nm）that met the needs of the experiment.Subsequently,the inner surface of the nanopore is sequentially subjected to hydrophilic treatment,as well as modification of sulfhydryl groups,gold nanoparticle and cAMP-specific antibodies,to functionalize the inner surface of the nanopore.When cAMP molecules are present in the system,cAMP will pass through the nanopore and bind to the cAMP-specific antibody on the nanopore wall.This process will cause the charge of the nanopore wall to change,which is shown as an increase in current under negative voltage on the I-V curve.As the concentration of cAMP increases,the current under negative voltage also gradually increases.And we further confirmed that the materials modified in each step of the nanopore wall,other antibodies,and other molecules similar in structure to cAMP can not cause changes in the ion rectification phenomenon.Therefore,the method we constructed is expected to help further detect and understand the signal transduction mechanism of small molecule second messengers in cells.（2）Glass nanopore sensor for the signal study of long-chain DNA translocation in different conformations.Nowadays,due to industrial development and too frequent human activities,many heavy metal ions are enriched in herbs,animals and humans,which seriously affects agriculture,aquaculture and public health.Some heavy metal ions can induce a specific G-quadruplex to undergo conformational changes.Therefore,the detection of heavy metal ions can be achieved by studying the formation of the G-quadruplex structure.Pb²⁺is a toxic heavy metal,which has profound significance for the specific and sensitive detection of Pb²⁺.In Chapter 3,we used the designed glass nanopore sensor and DNA molecule,combined with the resistive-pulse sensing,to study the signal of long-strand DNA translocation induced by Pb²⁺.We first silanized the inner surface of the glass nanopores（with a diameter of about 30 nm）prepared with a laser-based puller to improve the signal-to-noise ratio.When Pb²⁺is present in the system,the DNA chain regulated by it will change from a straight chain to a G-quadruplex structure.By analyzing the different resistance pulse signals generated by different conformations of DNA,the purpose of distinguishing different conformations of DNA can be achieved.The method designed in this chapter is expected to provide a new perspective for monitoring the signal of molecular translocation in nanopores,and is beneficial to further research on the signals of molecular conformation changes caused by other heavy metal ions.