Electrochemical Biosensor For Determination Of TP53 Gene And ATP Based On Gold Microelectrode

Cell is the basic unit of organism structure and functions.Analysis of genes,small molecules and proteins in single cell is helpful to understand cell metabolism,signal transduction and other phenomenon.It is of great significance to reveal the law of life activity and meaning for early diagnosis,prevention and treatment of tumor diseases.Therefore,to establish a simple,sensitive and rapid detection of genes,small molecules and other substances in single cell has become the important research part of life analysis.In recent years,a variety of single cell analysis methods and techniques have been developed for the detection of intracellular contents?such as protein,enzyme,and small molecules?or the monitoring of physical properties?such as morphology,and mitochondrial membrane potential?of single cells,such as microanalysis and microoperation,flow cytometry,fluorescence,optical tweezers,live cell probes,digital imaging and their related combination techniques.Some traditional methods have the disadvantages of large sampling volume,long analysis time and low sensitivity.so it is difficult to apply in single cell analysis,and the detection method of obtaining biomolecular chemical information in single cell is limited.Attribute to its simple equipment,the electrochemical method does not need to use probe to mark the target,which greatly reduces the disturbance to the normal physiological function of the cell.At the same time,single cell electrochemical analysis does not need to achieve high spatial resolution at the cost of time resolution,which is due to the fact that the collection of electrochemical signals does not need to be limited by the optical diffraction limit.The rapid development of preparation of microelectrode and nano electrochemical theory,greatly improve the detection limit,specificity and electrochemical measurement resolution.Therefore,the electrochemical method has emerged as an increasing important method for fundamental studies in single cell analysis.The aim of the present work is to develop novel electrochemical biosensors based on gold microelectrode to detect TP53 gene and continuously monitor ATP fluctuation at subcellular level.The main contents of the study are as follows:1.TP53 gene is the important tumor suppressor genes.The detection of TP53gene is important for early diagnosis and prognosis of tumor diseases.We designed a novel electrochemical sensor based on gold microelectrode for the detection of TP53genes.Firstly,three kinds of gold microelectrodes were prepared at the tip diameter of220 nm,120 nm,and 80 nm respectively,and were characterized by electrochemical method.Then TP53 gene capture probe with a thiol group at 3?-terminal was fixed onto the surface of the gold microelectrode through sulfhydryl self-assembly,and TP53gene capture probe labeled with ferrocene?Fc?at 5?-terminal was used to detect target TP53 genes.When no target TP53 gene,the Fc was far away from the electrode surface and the current signal was very small;when adding the target TP53 gene,the fixed single-stranded gene capture probe on the electrode hybridized with the target TP53 gene and formed a double-stranded DNA?dsDNA?,Fc was near the electrode surface,and the current signal increased.The detection limit of target TP53 gene was0.064 nmol/L.The target TP53 gene in cell extract was detected by this sensor,and the target TP53 gene in human serum was determined by the standard addition recovery.This work demonstrated that the prepared electrochemical biosensor based on gold microelectrode offers a great promising approach for sensitive detection of TP53genes.2.Monitoring the changes of adenosine triphosphate?ATP?at the subcellular level is important for studying cell energy metabolism.However,it is difficult for traditional ATP detection methods to provide information on the differences of ATP levels in different regions of cell.A real-time analysis method of ATP distribution and content in single cell was established in this study.A gold nanoelectrode with a diameter of120 nm was fabricated and ferrocene?Fc?-labeled anti-ATP aptamer was self-assembled onto the nanoelectrode surface to form nanoaptasensor.In the presence of target ATP,the Fc-ATP aptamer bound with two ATP units to form an ATP-aptamer conjugation that could fold into a stable framework composed of two stacked G-quartets,resulting in the close proximity of Fc to the surface of the microelectrode and then in an increased electrochemical oxidation current of Fc.ATP can be sensitively detected with a detection limit of 26?mol/L within 2 min.The ATP fluctuation at subcellular level was monitored continuously under starvation and Ca²⁺induction.This work demonstrates that the nanoaptasensor is a useful tool for investigating ATP-relevant biological processes via electrochemical method with high sensitivity,selectivity and spatial resolution.