Construction And Application Of High Sensitive Electrochemical Aptasensor Based On Signal Amplification Strategies

With the development of society and the improvement of human living standards,people are paying more and more attention to life and health,and the requirements for the detection of major disease markers are increasing.Highly sensitive detection of biomarkers in blood is key not only to find cancer at an early stage but also to help clinicians to decide the best treatment plan and to find how well treatment is working.However,these markers are mainly proteins present in low concentration levels in a high protein content medium,which hinders their detection.So it is necessary to develop a method which is simple,sensitive and selective for the detection of biomarkers.Electrochemical sensors,especially electrochemical nucleic acid aptamer sensors,have attracted much attention due to their high specificity and sensitivity,low cost,and wide application range.However,the traditional electrochemical method is difficult in the detection of low concentration.To quantify the small changes in clinically validated biomarkers,signal amplification strategies provide a new thought for improving the sensitivity of biosensor.In this dissertation,we developed three electrochemical and electrochemiluminescence aptasensors for the detection of the tumor marker MUC1and DNA oxidation damage marker 8-OH-dG,respectively.The sensitivity of the aptasensors was greatly improved due to the introduction of various signal amplification strategies such as catalytic performance of functional nanomaterials and isothermal nucleic acid amplification technology.The main content of the thesis includes the following three parts:1.A sensitive electrochemical aptasensor for Mucin 1 detection based on catalytic hairpin assembly coupled with PtPd NPs peroxidase-like activityIn this work,we prepared PtPd bimetallic nanoparticles with peroxidase-like properties.The nanoparticles have a strong ability to catalyze the oxidation of TMB by H₂O₂.In the design of the sensor,we introduced three hairpin DNAs—HP1,HP2and PtPd NPs-labeled HP3.Based on the ingenious design of the three hairpin DNAs,a highly sensitive electrochemical aptasensor for the detection ofMUC1 was constructed based on target-induced catalytic hairpin self-assembly.First,the specific binding of the target MUC1 with HP1 containing the aptamer sequence broke the hairpin structure of HP1,and then triggered the catalytic hairpin self-assembly of HP2and PtPd NPs-labeled HP3 on the electrode surface.In this process,the MUC1-HP1composite probe was released again through the chain substitution reaction,which triggered the next round of catalytic hairpin self-assembly,thereby achieving signal amplification.Based on the catalytic effect of PtPd NPs on the H₂O₂oxidation of TMB,MUC1 can be detected by measuring the current generated by the reaction.This aptasensor realized the recycling of the target through the catalytic hairpin self-assembly and chain substitution reaction.Combining catalytic hairpin self-assembly with the high catalytic activity of PtPd bimetal mimic oxidase,a new method for the sensitive detection ofMUC1 was developed.The detection limit of this sensor for MUC1 reached 16 fg/mL,which was lower than some reported electrochemical detection results.This design overcomes the shortcomings of traditional protein detection by protein conversion through enzyme cleavage or polymerization.In addition,compared with natural enzyme molecules,nanoperoxidase has higher sensitivity,better stability,and lower cost.This article provides a new idea for constructing simple,enzyme-free electrochemical sensors for protein detection.2.Ultrasensitive electrochemiluminescence aptasensor for 8-OH-2′-dG detection based on target-induced multi-DNA release and nicking enzyme amplification strategyIn this work,we developed an‘signal-on”ECL aptasensor for 8-OH-dG detection based on the structure-switching of Fc-labeled hairpin DNA（Fc-HP）immobilized on gold electrode.Unlike most electrochemical aptasensors prepared by directly immobilizing aptamer on the electrode surface,we firstly connected8-OH-dG aptamer with magnetic beads by amide bond.Then every aptamer hybridized with three kinds of short DNA strands（P1,P2,P3）,which consisted of two parts:one complementary to aptamer and another to Fc-HP.In the presence of8-OH-dG,three kinds of short DNAs were released again due to the affinity between aptamer and target.After magnetic separation,the released short DNAs were left in the supernatant.In the next procedure,the released short DNAs hybridized with Fc-HP immobilized on gold electrode to form double DNA strands.In the presence of nicking endonuclease（Nt.AlwI）,Fc-HP would be cleaved into two parts and one part containing Fc would leave the surface of electrode.Meanwhile,three kinds of short DNAs were released and then hybridized with another Fc-HP to initiate a new cycle of nicking endonuclease cleavage reaction.Because of nicking endonuclease-assisted recycling amplification,more Fc would leave the surface of the gold electrode.Accordingly,the ECL intensity of Ru（bpy）₃²⁺/TPA system would be enhanced greatly and the detection sensitivity would be improved.The fabricated aptasensor possessed two merits:First,every long aptamer hybridized with three kinds of short DNAs,one biological binding event would cause the release of three kinds of DNAs simultaneously,and as a result,triple the signal would be amplified.Second,nicking endonuclease-assisted amplification strategy realized the recycling of three kinds of short DNAs,which greatly improved the detection sensitivity.With such design,the ECL aptasensor achieved an ultrasensitive response to 8-OH-dG,indicating a promising potential for evaluation of oxidative DNA damage.3.Free-enzyme and triple-amplified electrochemical sensing of8-hydroxy-2’-deoxyguanosine by three kinds of short pDNAs-driven catalyzed hairpin assembly following with hybridization chain reactionBased on the second experiment,we used the magnetically separated pDNAs for the preparation of electrochemical sensors.The released three kinds of pDNAs initiate the catalyzed hairpin assembly（CHA）between HP1 and HP2 by hybridizing with HP1 immobilized on gold electrode.Meanwhile,the pDNAs are released again by strand displacement reaction.The released p DNA would successively participate in the next CHA process.Following that,the newly exposed HP2 fragment could launch HCR between HP3 and HP4 to form a long dsDNA structure.In this work,we used Ru（NH₃）₆³⁺as the electrochemical signal marker.There is an electrostatic attraction between the cationic Ru（NH₃）₆³⁺marker and the anionic DNA phosphate backbone.Thus Ru（NH₃）₆³⁺can be electrostatically adsorbed onto the dsDNA structure and the concentration of 8-OH-dG could be indirectly obtained by measuring the redox currents of Ru（NH₃）₆³⁺.By integrating triple signal amplification strategies,ultrasensitive detection toward 8-OH-dG could be achieved..In this work,triple signal amplification strategies were achieved without any enzyme,which,provides a new design idea for the detection of small molecules and other targets.The linear range of the sensor for 8-OH-dG detection is 100 fM-10 nM,and the detection limit is 24.34fM.It is used for the detection of 8-OH-dG in urine samples with satisfactory results.