| Electrochemiluminescence（ECL）has emerged as an ideal choice for detecting various biomolecules such as proteins,enzymes,and nucleic acids due to the combination of electrochemical analysis and chemiluminescence techniques,providing the unique advantages including low background,good reproducibility,wide dynamic range,high sensitivity and good controllability.However,there are still some challenges and limitations in the application of ECL technology,including the following two main aspects:（1）how to develop novel ECL nanomaterials with good dispersibility and stability to overcome the low luminescence efficiency caused by unstable free radicals;（2）how to achieve efficient conversion and signal amplification of low-abundance proteinases in complex biological systems to improve the detection range and reduce the background signal.Based on this,this study used various functionalized pyrene compounds as ECL luminophores,and combined with several improved nucleic acid amplification techniques to construct a highly stable and sensitive ECL sensing platform for ultra-sensitive detection of APE1 activity.In general,the research work of this paper was mainly divided into the following parts:1.Ag@Pyc nanoapsules as electrochemiluminescence emitters for an ultrasensitive assay of the APE1 activity.Polycyclic aromatic hydrocarbons（PAHs）are ECL emitters with great potential for application due to their high fluorescence quantum yield,good photochemical stability,and excellent photoelectric properties.However,the low solubility in aqueous solutions and unstable free radicals of PAHs result in weak ECL signals,which severely limits their further use in ECL biosensing.In this study,we developed a novel self-enhanced Ag@pyrenecarboxaldehyde nanocapsules（Ag@Pyc nanocapsules）as the ECL emitter to constructed an ultra-sensitive ECL biosensor platform for detecting human apurinic/apyrimidinic endonuclease 1（APE1）activity.Notably,Ag@Pyc nanocapsules have good hydrophilicity and stability,and Ag nanoparticles were attached to the surface of Pyc nanocapsules as co-reactants to significantly promote the production of the reaction intermediate sulfate radical anion(SO4·-)from the co-reactant persulfate ion(S2O82-),which interacted with Pyc nanocapsules to generate the strong ECL response.In addition,with the aid of APE1-triggered 3D DNA nanomachine,trace target could be converted into a large number of mimic targets（MTs）,which were positively correlated with the activity of APE1.Consequently,the proposed ECL biosensor realized an ultrasensitive detection of APE1 activity with an exceptional linear working range from5×10-10 to 5×10-4 U/μL and a lower limit of detection of 1.36×10-11 U/μL.This strategy provided a new approach to construct an efficient ternary system for the detection of biomolecules and early diagnosis of diseases.2.An ultrasensitive ECL biosening platform based on Pyc@GO nanosheets and target-triggered the recyclable cascade system for APE1 activity detection.Most organic nanocrystals experience quenching effects caused by aggregation in aqueous solutions.It is because the planar structure of PAHs is usuallyπ-πstacked,leading to severe internal filtering effects and excessive inactive emitters,which limits the ECL efficiency of the emitter.Herein,Pyrenecarboxaldehyde@Graphene Oxide（Pyc@GO）sheets with highly efficient electrochemiluminescence（ECL）as an emitter was prepared to develop a neoteric ECL biosensing platform for ultrasensitive assessment of human apurinic/apyrimidinic endonuclease1（APE1）activity.It is noteworthy that the Pyrenecarboxaldehyde（Pyc）molecules form stable polar functional groups on the surface of oxidized Graphene Oxide（GO）sheets through noncovalentπ-πstacking mechanisms,which enabled better dispersion of Pyc molecules on the GO surface and avoided their re-stacking.Compared with the tightly packed PAHs nanocrystals,Pyc@GO nanosheets significantly reduced internal filtering effects and diminished non-activated emitters to improve ECL efficiency and achieve strong ECL emission.Furthermore,the APE1-activated initiators could trigger the recyclable cascade amplified system based on the synergistic cross-activation between catalyzed hairpin assembly（CHA）and DNAzyme,resulting in improved signal amplification ability.Consequently,the proposed ECL biosensing platform for the detection of APE1 activity displayed exceptional sensitivity with a low detection limit of 4.6×10-9 U/m L ranging from 10-8 to 10-2 U/m L.Therefore,the developed strategy provides innovative idea and approach for the application of ECL techniques in bioanalysis.3.Pyc@γ-CD as ECL emitters combined with AND logic gate system for dual tumour markers detection.The signal transduction of proximity ligation assays relies on the conformational changes induced by protein binding.However,the exposed toehold of the affinity probe as a single strand lead to some nonspecific bindings and generate false-positive values.In this study,we designed a hairpin-lock DNA proximity circuit triggered by dual targets,combined with the Pyrenecarboxaldehyde@γ-cyclodextrin（Pyc@γ-CD）as an ECL emitter,to construct a dual-input regulated“AND”logic circuit based on a single ECL signal output for the detection of dual tumor markers.The well-designed hairpin-lock effectively suppressed the signal leakage from the circuit and reduced the background signal,which improved the detection sensitivity and accuracy of each target.Moreover,benefiting from host-guest recognition effects,the well-ordered assembly of Pyc molecule in specific micro/nano spaces effectively reduced the vibration and rotation of luminophores,suppressing non-radiative relaxation and achieving significantly enhanced ECL response.Therefore,the constructed ECL biosensor provides a new approach and direction for the efficient and sensitive detection of dual tumor markers,and this strategy is expected to play an critical role in the screening,differentiation and accurate diagnosis of tumor cells. |
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