Construction Of New Electrochemiluminescence Sensing Method Based On Resonance Energy Transfer In G-C₃N₄-based Nanocomposites And Its Applications

At the beginning of 2020,a sudden outbreak of pneumonia spread throughout the world,which brought great threats to people's life.People's understanding to life health and safety becomes unprecedented depth and attention.The development of life sciences,human health,and national economic and society makes the increasing challenge and demands for analytical chemistry.Life analytical chemistry has shown increasing significance and value.Electrochemiluminescence?ECL?,a favorable analytical technique combining electrochemistry and chemiluminescence,has attracted attention of the researchers,with the features of high sensitivity,wide linear range,and fast analysis speed.The combination of ECL active materials and new signal amplification strategies shows great development prospects and significance.In this paper,a series of g-C₃N₄-based nanocomposites with excellent ECL properties(such as g-C₃N₄-MnO₂,g-C₃N₄-Ag,g-C₃N₄-Cd²⁺,g-C₃N₄-CoOOH nanocomposites)were synthesized.Innovatively integrating with resonance energy transfer?RET?,etching reaction,redox cycling amplification,a series of new ECL sensing platforms were designed,realizing the sensitive and accurate detection of bioactive molecules.The main innovations and research contents are as follows.1.Electrochemiluminescence energy resonance transfer in 2D/2D heterostructured g-C₃N₄/MnO₂ for glutathione detectionThe energy transfer efficiency,strongly depending on the distance of donor–acceptor pair,is always a crucial factor for the construction of elegant ECL-RET-based biosensors.In this work,a novel and efficient ECL-RET in 2D/2D heterostructured g-C₃N₄/MnO₂ was developed using g-C₃N₄ nanosheets?g-C₃N₄ NSs?as energy donor and MnO₂ nanosheets?MnO₂ NSs?as energy acceptor.In this system,MnO₂ NSs in-situ grew on g-C₃N₄ NSs to form the 2D/2D heterostructure,greatly shortening the distance of the donor–acceptor pair?g-C₃N₄–MnO₂?and thus greatly enhancing the RET efficiency.To demonstrate the performance of the system,a signal“off-on”ECL sensor was designed for glutathione?GSH?analysis.In the absence of GSH,MnO₂significantly quenched the ECL intensity of g-C₃N₄ owing to ECL-RET in this 2D/2D g-C₃N₄/MnO₂ heterostructure?ECL signal“off”?.Upon the addition of GSH,MnO₂was reduced to Mn²⁺by GSH and g-C₃N₄ was released from the heterostructured g-C₃N₄/MnO₂,generating a recovery of ECL intensity?ECL signal“on”?.Under the optimal conditions,the designed ECL-RET signal“off-on”sensor realized the sensitive detection of GSH ranged from 0.2?mol/L–100.0?mol/L with the detection limit?LOD?of 0.05?mol/L.Furthermore,the as-prepared ECL-RET sensor exhibits good performance in the determination of GSH in human serum samples,the recoveries varied in the range of 87.0–112.0%and the relative standard deviations?RSDs?were no more than 6.6%.The ECL-RET in 2D/2D heterostructure provides an ingenious way for the exploitation of novel ECL biosensing systems.2.Highly efficient resonance energy transfer in g-C₃N₄-Ag nanostructure:Proof-of-concept toward sensitive split-type electrochemiluminescence immunoassayA high-efficient ECL-RET mode between luminescent materials and noble metal nanoparticles was highlighted,in which luminescent materials?g-C₃N₄ nanosheets as the energy donor?and noble metal nanoparticles?Ag nanoparticles as the energy acceptor?were innovatively combined into one nanostructure,greatly reducing the energy loss and effectively promoting the RET efficiency.To prove the concept of the RET mode in ECL bioanalysis,a split-type ECL immunosensor was developed using interleukin-6?IL-6?as a model.In the presence of IL-6,a sandwich immunoreaction occurred in anti-IL-6 antibody-coated 96-well plate by using glucose oxidase and anti-IL-6 antibody-conjugating silicon dioxide nanoparticles?anti-IL-6-SiO₂-GOx?as signal label.The carried GOx oxidized glucose to generate H₂O₂,which etched Ag nanoparticles in g-C₃N₄-Ag nanostructure,thereby destroying the ECL-RET between g-C₃N₄ and Ag and thus recovering the ECL intensity.The high-efficient ECL-RET in one nanostructure and GOx-induced H₂O₂ etching modulation in this proposed system endowed the high sensitivity of the immunoassay toward IL-6 with the dynamic working range of 1.0×10^-13–1.0×10^-8 g/mL and the LOD of 3.2×10^-14 g/mL.It's believed that this ingenious combination of g-C₃N₄ and Ag into one nanostructure provides a new perspective to expand the application of ECL-RET of luminescent materials and noble metal nanoparticles.3.Dual-wavelength electrochemiluminescence ratiometry based on Cd²⁺doped g-C₃N₄ nanosheets for H₂SECL ratiometric analysis is a method based on the ratio of two ECL signals,which could effectively avoid the false negative or positive errors,thus improve the sensitivity and accuracy of target detection.Yet,the luminescence intensity and wavelength of ECL materials limit the application of the ratiometric analysis.Exploring the suitable materials for ratiometric ECL still remains a big challenge.In this work,a dual-wavelength ratiometric ECL-RET sensing platform was constructed based on the Cd²⁺-doped g-C₃N₄ NSs for H₂S sensitive detection.In this approach,Cd²⁺-doped g-C₃N₄ NSs exhibited a strong ECL emission at 435 nm.Upon the presence of H₂S,CdS was formed in-situ on g-C₃N₄ NSs by the absorption of S^2-ion and Cd²⁺ion,generating another ECL emission at 515 nm.Furthermore,the overlapping of absorption spectrum of the formed CdS and ECL emission spectrum of g-C₃N₄ NSs led to the quench of ECL intensity from g-C₃N₄ at 435 nm.Based on the ECL quenching at 435 nm and increasing at 515 nm,the sensitive and accurate assay of H₂S was realized by measuring the ratio of ECL_{435 nm}/ECL_{515 nm}.The linear ranges were from 0.05?mol/L to 100.0?mol/L with the LOD of 0.02?mol/L.This work exploits the dual-wavelength ratiometric ECL-RET sensor based on the in-situ generation of the luminescent reagents,which provides a new direction for the development of ECL ratiometric sensing platform.4.Chemical-chemical redox cycling for advanced signal amplification:Toward sensitive myoglobin electrochemiluminescence bioanalysisThis work developed an elegant bridging of chemical-chemical?CC?redox cycle with high-efficient RET signal amplification mechanism for ultrasensitive split-type ECL immunoanalysis.RET donor g-C₃N₄ NSs and acceptor CoOOH were innovatively combined into one nanostructure to form g-C₃N₄-CoOOH nanomaterials,greatly reducing the energy loss and effectively promoting the RET efficiency.After a sandwich immunoassay,the signaling species AA was catalytic generated by alkaline phosphatase,triggering the etching reaction between AA and CoOOH.The introduction of tris?2-carboxyethyl?phosphine?TCEP?made the cycling regeneration of AA,thereby achieving the CC redox cycling and leading to novel signal amplification.Exemplified by myoglobin?Myo?as the target,CC redox cycle and high-efficient RET in one nanostructure realized the ultrasensitive ECL immunoassay with the dynamic working range of 5.0×10^-13–1.0×10^-7 g/mL and the LOD of 2.0×10^-13 g/mL.In addition,the designed method has been successfully applied to the determination of Myo in human serum samples.The results remained nearly consistent with the reference values of Xinyang Central Hospital with the recoveries of 90.0%to108.0%.This work firstly introduced CC redox cycle into the ECL-RET biosensing analysis.We envision that such a protocol will offer new ideas for the extended research of redox cycle,and provide a new perspective for the sensitive ECL biological analysis.