The Construction Of An Electrochemical Aptasensors Based On The Host-guest Recognition And Its Application In Thrombin Detection

Thrombin is an important serine protein in the human body that plays a key role in the pathological and physiological coagulation of diseases as diverse as thromboembolic disease,cancer and Alzheimer’s disease.Excessive thrombin concentration can cause blood clots,while the reduced amount of thrombin can cause bleeding.Therefore,it is very necessary to establish a sensitive and efficient detection method for the diagnosis of diseases caused by abnormal thrombin content.Electrochemical biosensor is composed of biometric identification materials and detection devices.It is sensitive to biological substances and can convert the concentration of detected substances into electrochemical signals to achieve the purpose of detection.Compared with other electrochemical sensors,due to its many advantages,such as high sensitivity,good selectivity,simple operation,low detection cost,and fast response speed,electrochemical biosensors have become more and more widely used.In recent years,aptamers have become a research focus for more and more people.It is a typical artificial single-stranded DNA or RNA,and its most notable feature is its high binding affinity and specificity.Aptamers can bind to various targets,such as heavy metal ions,proteins,viruses and cancer cells.They have the following advantages: rapid synthesis in vitro,easy modification,no impact on their biological activity,no immunogenicity,non-toxicity,high tissue permeability and high stability.For this reason,aptamers with good affinity and high specificity and electrochemical active substances are used as biological recognition elements.Combination of host-guest recognition and hairpin assembly catalysis for signal amplification,a highly sensitive and specific electrochemical aptasensor was constructed to achieve semi-quantitative or quantitative detection of low content thrombin.The experimental results show that the electrochemical aptasensor constructed in this paper has high sensitivity and selectivity for thrombin detection,and it has been successfully used for the rapid detection of thrombin in serum samples,indicating that the constructed aptasensor has potential clinical application value.The research work of this paper is mainly divided into the following two parts:(1)Based on the host-guest recognition between tetraferrocene and β-cyclodextrin(β-CD),a homogeneous electrochemical aptasensor was constructed and successfully applied to the rapid detection of thrombin.In this chapter,a novel ferrocene derivative,tetraferrocene,was synthesized,which contains four ferrocene units,and was modified at both ends of the double hairpin DNA probe to form a thrombin aptamer(TBA)with high signal acquisition function.Through the self-assembly method,the synthesized sulfhydryl β-cyclodextrin(SH-β-CD)is fixed on the surface of the gold electrode(Au E).In the absence of thrombin,the double-stem-loop structure of TBA prevents the tetraferrocene labeled at both ends from entering the β-CD cavity on the surface of the gold electrode.When TBA binds to thrombin,the stem loop structure of TBA opens and transforms into a special G-quadruplex structure.At this time,the tetraferrocene modified at both ends of TBA enters the cavity of β-CD.Therefore,the amplified current signal can be directly detected.The constructed aptasensor realizes the sensitive and rapid detection of thrombin,the linear range of 4 p M to 12.5 n M and a detection limit of 1.2 p M(S/N=3).(2)Based on the target-initiated catalytic hairpin assembly(CHA)technology and the dual signal amplification strategy of tetraferrocene and β-CD host-guest recognition,an electrochemical aptasensor was constructed for the rapid detection of thrombin.The aptasensor consists of two parts.One part is the detection substrate.The SH-β-CD is modified on the surface of the gold electrode to form the detection substrate,which can generate a stable current signal.The other part is the recognition probe.the other is the probe.The synthesized tetraferrocene was modified at the both ends of double hairpin probe,and a hairpin structure of the probe has thrombin specific recognition genes.One of the hairpin structures of the probe had a specific thrombin recognition nucleotide sequence.The target thrombin and TBA are specifically recognized.Though CHA reaction,thrombin is replaced and continues to catalyze the next reaction.Therefore,the low concentration of thrombin can obtain a double enhanced electrochemical response signal.The electrochemical aptasensor is based on CHA technology and hostguest recognition between tetraferrocene and β-CD,which significantly amplifies the electrochemical signal of thrombin and realizes the sensitive detection of thrombin.The aptasensor shows a good linear relationship in the range of 1 pm-3.125 n M,and the detection limit is 0.28 p M(S/N=3).In this paper,thrombin,which has been studied the most,was select as the analysis object.and two electrochemical aptasensors were constructed.The main advantages are as follows: 1)The constructed aptasensors are detected in a homogeneous solution,and there is no need to fix the aptamer probe on the electrode surface,which greatly reduces the steric hindrance between the aptamer probe and the target thrombin,and improves the recognition efficiency.2)The synthesized new electroactive substances can directly obtain electrochemical signals without additional substrates such as enzymes,simplifying experimental operations,reducing costs,and improving new electroactive substances for the development of sensors.3)The reaction system does not require the addition of complex nanomaterials and enzymes,and can specifically recognize the target thrombin in the presence of other interfering proteins.