Study On Highly Sensitive Homogeneous Electrochemical Aptasensors Based On Different Electrodes

In the traditional electrochemical aptasensors,the immobilization processes of bioprobes on the electrode surfaces were laborious and time-consuming.Moreover,the recognitions between aptamers and their targets occurred on the solution-electrode interfaces that would reduce their binding efficiency.To overcome these limitations,some immobilization-free systems were developed by utilizing indium tin oxide?ITO?electrode as the sensing platform.In these work,the biorecognition processes occurred in the homogeneous solutions and the signal outputs were relied on the diffusion currents of the electroactive molecules from solution phase to the electrode surface.However,these diffusion-mediated systems could not efficiently utilize the freely dispersed signal molecules in the whole system,which limited the sensitivity of the sensors.Also,the ITO electrode itself is difficult to amplify the electrochemical signals.Thus,it is still desirable to develop some convenient homogeneous platforms with effective signal output/amplification mechanisms.In this paper,different modified electrodes were employed as the novel homogeneous electrochemical sensing platforms.These platforms were not only easy to prepare,but also could electro-catalyze or enrich the signal molecules to effectively amplify the signals.By coupling with the RecJ_f exonuclease-assisted target recycling,the highly sensitive detections of thrombin?TB?were realized.The main works were as follows:?1?Study on“signal-off”homogeneous electrochemical aptasensors based on electro-catalytic amplification of graphene and exonuclease-assisted target recyclingIn this work,the reduced graphene oxide modified glassy carbon electrode?RGO/GCE?was employed as the sensing platform.A DNA hairpin probe?HP?was ingeniously designed,which contained the complementary sequence to thrombin-binding aptamer?TBA?in its 3?-protruding terminus.In the absence of the target TB,HP hybridized with TBA to form the HP-TBA duplex,which had the weak affinity to RGO/GCE.The resulted electrode showed a remarkable electrocatalytic current for uric acid?UA?oxidation(I_UA).Upon the target binding,TBA was dissociated from duplex and the TB-TBA complex was obtained.The released HP was strongly adsorbed on the RGO/GCE through?-?stacking interaction and covered its active surface.So that the UA signal was decreased.While RecJf exonuclease was further introduced,the TBA in the TB-TBA complex was digested.The liberated TB could combine with its aptamer in another HP-TBA duplex to initiate the next reaction cycle.Thus,the observed UA signal was further reduced.The detection of TB would be realized by monitoring this distinct change in I_UA.In the optimized experimental conditions,the developed aptasensor exhibited excellent analytical performance for TB,such as the wide linear range from 5 to 1000 fmol L^-1,the low detection limit of 0.86 f fmol L^-1?S/N=3?,the good reproducibility and stability,as well as the satisfied application in the serum samples.?2?Study on“signal-on”homogeneous electrochemical aptasensor based on electro-catalytic amplification of graphene and exonuclease-assisted target recyclingIn this work,the reduced graphene oxide modified glassy carbon electrode?RGO/GCE?was employed as the sensing platform.The Hairpin DNA was designed with a closed 5?terminus and a protruded 3?terminus.It contained the sequence of the thrombin-binding aptamer,and is named TBA.In the absence of TB,RecJ_f exonuclease could not recognize and digest TBA due to its closed 5?terminus.Via the protruded 3?terminus,TBA was adsorbed on the surface of RGO/GCE through?-?stacking interaction and covered the active surface of the electrode.Thus,a weak catalytic oxidation current of UA(I_UA)was observed.Upon the target binding,the TB-TBA complex was formed,which was hardly adsorbed on the surface of RGO/GCE.Therefore,the detected IUA is increased.While the RecJf exonuclease is further introduced,the TBA in the TB-TBA complex was digested.The released TB continued to combine with TBA to initiate the next reaction cycle,resulting in that more TBA was digested.Thus,less TBA could be adsorbed on the surface of RGO/GCE,leading to a futher increased I_UA.The detection of TB could be realized by monitoring the change of I_UA.In the optimized experimental conditions,the developed aptasensor exhibited excellent analytical performance for TB,such as the wide linear range from 1 to 1000 fmol L^-1,the low detection limit of 0.21 fmol L^-1?S/N=3?,the good reproducibility and stability,as well as the satisfied application in the serum samples.?3?Study on a ratiometric homogenous electrochemical aptasensor based on the competitive host-guest recognition of cyclodextrin and exonuclease-assisted target recyclingIn this work,a poly-?-cyclodextrin modified glassy carbon electrode?p?-CD/GCE?was employed as the sensing platform.The Hairpin DNA with a closed5?terminus was modified with rhodamine B?RB?in its protruded 3?terminus.It contained the sequence of the thrombin-binding aptamer,and was named RB-TBA.In the absence of TB,RecJ_f exonuclease could not recognize and digest RB-TBA due to its closed 5?terminus.Through the host-guest recognition between?-cyclodextrin and RB,RB-TBA was adsorbed on the surface of p?-CD/GCE and shield the 1-naphthol?1-Nap?away from the electrode.In this case,an intense current of RB?IRB?and a weak current of 1-Nap(I_Nap)were observed.Upon the target binding,the RB-TBA/TB complex was formed.Because of the steric hindrance of TB,RB hardly entered into the inner cavity of?-CD,leading to less RB-TBA on p?-CD/GCE.1-Nap could combine with the exposed?-CD.Thus,the detected I_RBB was decreased with the increased I_Nap.While the RecJ_f exonuclease was further introduced,the RB-TBA in the RB-TBA/TB complex was digested,and the TB and RB were released.The liberated TB continued to combine with RB-TBA to initiate the next reaction cycle.Thus,the number of RB-TBA on p?-CD/GCE was further reduced.Many free RB could enter into the the inner cavity of?-CD,but they were then replaced by 1-Nap.Therefore,the RB signal was further suppressed and 1-Nap signal was further enhanced.With the I_Nap/I_RB as the response signal,the detection of target could be realized.In the optimized experimental conditions,the developed aptasensor exhibited excellent analytical performance for TB,such as the wide linear range from0.5 to 1000 fmol L^-1,the low detection limit of 0.12 fmol L^-1?S/N=3?,the good reproducibility and stability,as well as the satisfied application in the serum samples.