Research On The New Electrochemiluminescence System Based On 3D G-C₃N₄ For Cancer Marker Detection

Electrochemiluminescence（ECL）biosensor is the combination of electrochemical methods,chemiluminescence methods,and sensor technology.It is efficient and sensitive,and has gradually become an important analytical tool in the fields of clinical detection,environmental detection and food analysis.The luminescene properties of ECL nanomaterials are the basis of ECL analysis technology,and finding new,efficient and green ECL nanomaterials is the focus of the research.At present,graphitic carbon nitride（g-C₃N₄）has attracted scholars’attention due to its low cost,low toxicity and excellent chemical stability.However,the luminescene efficiency of g-C₃N₄ is low,and the existing research focuses on two-dimensional g-C₃N₄,while 3D g-C₃N₄ is rarely studied.Because of the low intensity of annihilation ECL,the coreactant ECL is the focus of research.Among all kinds of coreactants,dissolved O₂ has the advantages of mild reaction,simple operation and good biocompatibility.However,compared with other coreactants,dissolved O₂ has low reaction activity and weak ability to promote the luminescence of ECL nanomaterials.Therefore,it is particularly important to build novel system which has high co-reaction efficiency with dissolved O₂ as the coreactant.In addition,in the aspect of analysis and biosensing,the sensitivity of the biosensor is its main performance,so it is very important to propose reasonable and effective amplification strategy.Based on above,this paper constructed new ECL systems based on 3D g-C₃N₄ as the ECL emitter and dissolved O₂ as the coreactant.At the same time,based on an efficient nucleic acid signal amplification strategy,the ultra-sensitive biosensors were constructed for the detection of cancer markers.The research works include the following parts:1.High efficiency electrochemiluminescence of 3D porous g-C₃N₄ with dissolved O₂ as co- reactant and its sensing application for ultrasensitive detection of microRNA in tumor cellsIn this work,the electrochemiluminescence（ECL）phenomenon of three-dimensional graphitic carbon nitride（3D g-C₃N₄）was reported.Firstly,the proposed 3D g-C₃N₄ possessed3D porous interconnected open-framework which enabled faster charge transport and efficient penetration of co-reactants due to“pore confinement effect”.Then,we found that the dissolved O₂ could serve as an excellent coreactant for cathodic ECL of 3D g-C₃N₄.And the high specific surface area was beneficial to better adsorbing and gathering of dissolved O₂ and reactive oxygen species（ROSs）,which made them full contact on the surface or inside of 3D g-C₃N₄,giving a more sufficient ECL reaction and higher ECL signal.Based on the proposed 3D g-C₃N₄-O₂ ECL system,a sensitive biosensor was constructed for microRNA-21（mi RNA-21）detection with assistance of 3D spherical tracks assisted 3D DNA walking machine,which exhibited superior performance for mi RNA-21 with detection limit of 0.22 f M.The proposed3D g-C₃N₄-O₂ ECL system with high ECL efficiency and the effective target conversion amplification strategies were beneficial to construct ultra-sensitive ECL sensing platform,which would be better applied to clinical bioanalysis.2.High-Density N Vacancy Induced Multi-Path Electrochemiluminescence Improvement of 3D g-C₃N₄-NV-dissolved O₂ System for ultra-sensitive Mi RNA-222 AnalysisUsing dissolved O₂ as the cathodic co-reactant of 3D g-C₃N₄ is a convenient method to improve the electrochemiluminescence（ECL）signal,but it still suffers the disadvantages of limited luminous efficiency of 3D g-C₃N₄ and low content,low reactivity and instability of dissolved O₂.Here,N vacancy with high density was firstly introduced into the structure of 3D g-C₃N₄（3D g-C₃N₄-NV）,which could conveniently realize multi-path ECL improvement by simultaneously solving the above shortcomings effectively.Specifically,N vacancy could change the electronic structure of 3D g-C₃N₄ to broaden its band gap,slower decay dynamics and accelerate electron transfer rate,obviously improving the luminous efficiency of 3D g-C₃N₄.Meanwhile,N vacancy made the excitation potential of 3D g-C₃N₄-NV shifted from-1.3 to-0.6 V,effectively weakening the electrode passivation which often occurred in g-C₃N₄ based ECL under high potential.Compared to the traditional N vacancy in 2D g-C₃N₄,the N vacancy in 3D g-C₃N₄ possessed higher density due to 3D porous structure,which had better regulation effect and obvious improvement for the ECL efficiency.Moreover,the adsorption capacity of3D g-C₃N₄-NV was obviously enhanced,which could make the dissolved O₂ enrich around 3D g-C₃N₄-NV.And massive active NV sites of 3D g-C₃N₄-NV could promote O₂ more efficiently convert to reactive oxygen species（ROSs）that were key intermediates in the ECL generation.Thus,the mentioned deficiencies of co-reactive dissolved O₂ were effectively improved to acquire an excellent ECL emission.Using the newly proposed 3D g-C₃N₄-NV-dissolved O₂system as ECL emitter,an ultra-sensitive target conversion biosensor was constructed for mi RNA-222 detection.DNA nanomachine containing multi-HCR derived from DNA nanocombs could more quickly and effectively convert quantitative targets into a large amount of output DNA.Furthermore,on the electrode,numerous ferrocene was loaded in DNA hydrogels to greatly reduce the background signal.The fabricated ECL biosensor exhibited satisfactory analytical performance for mi RNA-222 with the detection limit of 16.6 a M.The strategy achieved multi-path ECL improvement by introducing high density N vacancy simply in the 3D structure of g-C₃N₄ could open a new horizon for developing high performance ECL system.3.Excellent 3D Th_ing-g-C₃N₄-Dissolved O₂ ECL System Performance Achieved by Doping Thiophene Ring for Mi RNA-221 AssayDue to its low luminous efficiency,the application of g-C₃N₄ in the field of ECL is limited.In this work,we proposed to dope thiophene ring into 3D g-C₃N₄ to build a high-speed charge transfer network to promote 3D g-C₃N₄ intramolecular charge transfer,realizing the efficient luminescence of 3D g-C₃N₄ with dissolved O₂ as coreactant.Based on the 3D Th_ing-g-C₃N₄-dissolved O₂ system,the target-like substance obtained through the efficient HCR reaction could trigger the HCR reaction again to realize the effective amplification of the target.The constructed biosensor had excellent detection performance for mi RNA-221 with a detection limit of 5.25 a M.In conclusion,the 3D Th_ing-g-C₃N₄ with high ECL efficiency broadened the application of g-C₃N₄ in the field of ECL,and the biosensor construction strategy opened up a new way for the ECL biosensor platform for early cancer diagnosis.