The Construction Of Electrochemiluminescence Aptasensor And Its Application In Biomarkers Assay

Electrochemiluminescence(ECL),which combines the traditional electrochemical and chemiluminescence,is a kind of chemiluminescence driven by electrochemical reaction.It has wide dynamic range,good sensitivity,near-zero background and high time/spatial-resolution.As a strong analytical technology,it is widely used in many flied which could range from fundamental studies to practical applications,such as biosensing and imaging,environmental monitoring and clinical detection.Some nucleic acid amplification strategies are often introduced in order to amplify signal and further achieve higher sensitivity for the detection of trace target.The nucleic acid amplification strategies are mainly divided into enzyme-assisted cyclic amplification(such as polymerase chain reaction)and enzyme-free cyclic amplification(such as strand displacement amplification),which can be widely used in the construction of biosensor.Biomarkers of disease exist at very low level in the organism even though they are some overexpressed proteins mostly.It can be used in early screening of diseases or as a monitoring indicator in the period of diagnosis and treatment.Therefore,the development of a rapid and sensitive method for the detection of disease biomarkers is of great significance in clinical detection.The study includes following three parts main parts:(1)The ECL sensor based on?-CD/g-C₃N₄ composite for the detection of PDGF-BBThe ECL aptasensor was fabricated to achieve the detection of platelet derived growth factor BB(PDGF-BB)based on?-cyclodextrin/graphitic carbon nitride composite(?-CD/g-C₃N₄),which was prepared by non-covalent modification.?-CD/g-C₃N₄ can be immobilized on the electrode by the excellent film-forming ability of chitosan.The adamantane-labeled DNA(Ad-DNA)and the ferrocene-labeled aptamer(Fc-DNA)was gradually modified on the electrode surface via host-guest recognition and complementary base pairing.The Fc molecule could effectively quench the ECL signal of g-C₃N₄ which was probably caused by both energy transfer and photo-excited electron transfer process.There was a stronger affinity between the aptamer and the target than the hydrogen bond of double-stranded DNA structure.When the target PDGF-BB was present,it interacted with Fc-DNA and then moved away from the electrode surface,leading to the recovery of ECL signal to some extent.There was a linear response between the increased value of ECL intensity(?I)and the logarithm of PDGF-BB concentration from 0.5 pg/m L–0.5?g/m L,the limit of detection was 0.26 pg/m L,which demonstrated that the high sensitivity of this aptasensor.Meanwhile,it could be used for the detection of PDGF-BB in serum samples.(2)The ECL sensor based on resonance energy transfer for the detection of ThrombinThe ECL aptasensor was constructed based on the strategy of resonance energy transfer(RET)between PFBT polymer dots(PFBT Pdots)and black hole quencher(BHQ)molecule for sensitive assay of thrombin(Tb).PFBT Pdots,as a novel ECL lumiphore,was synthesized by simple nanoprecipitation in aqueous solution.The modified electrode was prepared by specific binding of streptavidin and biotin.Meanwhile,the exonuclease III-assisted enzymatic could improve efficiency of ECL-RET from lumiphore PFBT Pdots to BHQ molecule,leading to more decrease of ECL signal,which could effectively improve sensitivity further.The results showed that there was a good response in the range of 10 pg/m L–100 ng/m L for the detection of Tb,and the limit of detection was 3.9 pg/m L.The obtained sensor had high sensitivity,strong anti-interference ability and good stability,it had also been applied to detect the concentration of Tb in human serum with satisfied results,which provided potential application in future.(3)The ECL sensor based on strategy of dual signal amplification for the detection of Mucin 1The ECL biosensor was fabricated based on dual signal amplification strategy of endonuclease Nt.Bsm AI-assisted cyclic amplification and strand displacement amplification to achieve the sensitive detection of mucin 1(MUC1).Au-Limunol nanoparticles(Au-Luminol NPs),as the lumiphore,which was prepared by the directly reduction of HAu Cl₄ with luminol.A significant ECL response can be produced under the presence of the co-reactant hydrogen peroxide.A large amount of intermediate DNA(i-DNA)could be produced by restriction endonuclease Nt.Bsm AI-assisted aptamer-target complex circulation in advance.The i-DNA would open the hairpin structure of capture DNA on the electrode surface.Subsequently,the DNA labeled by lumiphore Au-Luminol NPs was modified on the electrode surface through strand displacement amplification strategy to realize the recycling of the i-DNA.Thereby realizing the construction of the ECL biosensor and effectively improving the sensitivity.The results illustrated that there was a positively linear response between the ECL intensity and the logarithm of target MUC1 concentration at 1 pg/m L-5 ng/m L,the limit of detection was 0.71pg/m L.The constructed biosensor had high sensitivity and good selectivity,it could be applied to the detection of MUC1 in human serum with the recovery ranging from 95%to 104%.