Construction Of Electrochemical Impedance Aptasensor And Its Application In Assay Of Disease Markers

Protein is an extremely important polymer organic substance that constitutes human cells and tissues.In the life activities of the human body,normal physiological functions cannot be performed without proteins.In clinical analysis,disease markers refer to specific biomarkers related to the disease.More and more studies have shown that the occurrence and development of many diseases are closely related to the abnormal expression of some specific proteins,and in the early stage of disease,the content of these disease-specific proteins is extremely low,which is difficult to distinguish from other proteins with high content in the body.Therefore,achieving highly sensitive and selective detection of protein-based disease markers is of great significance for the early diagnosis and treatment of diseases.The electrochemical impedance sensor has the advantages of simple structure,fast response,and no signal labeling;The nucleic acid aptamer is obtained by in vitro screening,is easy to be chemically modified,and has a strong ability to specifically recognize the target.If the nucleic acid aptamer is introduced into the electrochemical impedance sensor,combining the advantages of the two,constructing the electrochemical impedance aptasensor will further improve the selectivity of the electrochemical sensor.The traditional electrochemical impedance aptasensor has the disadvantages that the signal change before and after capturing the target is small and the sensing mode is simple.In this paper,based on the target-induced dual exonuclease inhibition strategy and target-induced single exonuclease inhibition strategy,two electrochemical impedance aptasensors for highly sensitive detection of human interferon-? and thrombin were constructed,respectively,which improved the detection sensitivity,reduced the signal labeling process and the use of nucleic acid tool enzymes,reduced the experimental cost,and realized highly sensitive detection of protein disease markers.The main content of the paper includes the following two aspects:(1)Based on the target-induced dual exonuclease inhibition strategy,an electrochemical impedance aptasensor for highly sensitive detection of human interferon-?(IFN-?)was constructed.We designed a DNA hairpin modified on the gold electrode(loop was the aptamer of the IFN-?,hereafter referred to as aptamer).In the absence of IFN-?,Exonuclease III(Exo III)and Exonuclease I(Exo I)digested the double-stranded and single stranded parts of the aptamer respectively to obtain single nucleotides,thus generated smaller impedance value on the electrode surface.In the presence of the target,the function of Exo III was greatly inhibited by the binding of the aptamer with the target and stopped after cutting three bases of the hairpin DNA.The formed IFN-?-aptamer conjugate could not be digested by Exo I but increased the electron transfer resistance,generating larger impedance on the electrode surface.Under optimal experimental conditions,the impedance change had a good linear relationship with the logarithm of IFN-? concentration in the range of 1 p M to 50 n M,and the detection limit was0.7 p M.This aptasensor had the advantages of high selectivity,reproducibility,low cost,and could be used for detection IFN-? in actual samples.It is expected to provide an effective method for the early diagnosis of tuberculosis.(2)Based on the target-induced single exonuclease inhibition strategy,an electrochemical impedance aptasensor for highly sensitive detection of thrombin(Tb)was constructed.The 15-base aptamer TBA15 was modified on the gold electrode,when there was no target,TBA15 could not be combined with the 29-base aptamer TBA29.However,it could be digested into single nucleotides by Exo I,causing the electrochemical impedance on the electrode surface was relatively smaller.In the presence of Tb,the TBA15-Tb-TBA29 conjugate was formed between Tb,TBA15 and TBA29,which inhibited the digestion of Exo I.Thus,huger electrochemical impedance was generated on the electrode surface.Under the optimized experimental conditions,impedance value and the logarithm of the Tb concentration showed a good linear relationship in the range of 1 ng/m L to 1000 ng/m L,and the detection limit was0.18 ng/m L(about 5.04 p M).The strategy that target-induced single exonuclease inhibition could quickly,easily and sensitively detect the target concentration.