High-Efficiency Electrochemiluminescent Nitrogen-Doped Carbon Dots:Synthsis And Bioanalytical Applications

Carbon dots?CDs?is a new type of electrochemiluminescence?ECL?reagent with outstanding advantages of low toxicity,low cost and good biocompatibility,which has an extensive application prospect in the field of ECL bioanalysis.However,the ECL performance of CDs is still unsatisfactory due to the main disadvangtages of high ECL excitation potential and low ECL efficiency,which significantly prevents them being widely applicated.Nitrogen doping is one of the conventional methods to improve the ECL performance of CDs,which retains the high stability and low cost of CDs to the greatest extent.Unfortunately,the role of doped nitrogen atoms in the ECL process of CDs has still not been clarified so far,which leads to the situation that the improvement of ECL performance of CDs through nitrogen doping is guideless and the results caused by nitrogen doping are often uncontrollable.In this paper,a series of nitrogen-doped CDs with different structures were prepared by using different nitrogen sources.By comparing the ECL properties of different CDs,the general rules about the relationship between the structure of doped nitrogen atoms and the performance of ECL were summarized.Meanwhile,with the aid of quantum chemistry tools,the principle of doped nitrogen atoms determining the optical and electrochemical properties of CDs was revealed.According to the above rules and principles,the structure and preparation protocol of nitrogen-doped CDs were designed specifically to obtain novel nitrogen-doped CDs with low excitation potential and high ECL efficiency by decreasing the intrinsic redox potential and promoting the coreaction efficiency and excited state quantum yield of CDs.Finally,ECL biosensors were constructed and applied in vitro detection of biomarkers and in situ analysis of living cells combining the prepared CDs with appropriate biometrics and signal amplification strategies.The specific research work is divided into the following parts:1.Anodic electrochemiluminescence of carbon dots promoted by nitrogen doping and application to rapid cancer cell detectionVIn the living cell analysis,the biotoxicity of ECL emitters will seriously interfere with the normal life activities of living cells,thus reducing the accuracy of analysis.Therefore,the development of a new ECL system with good biocompatibility is very important for the application of ECL methodology in living cell analysis.CDs,a kind of optical emitters,have excellent biocompatibility.However,it also has the problems of low ECL efficiency and high excitation potential.In this work,according to the general rule of nitrogen doping to reduce the oxidation potential of carbon materials,the feasibility of reducing the ECL excitation potential of CDs by nitrogen doping has been studied.Hydrazide coupled CDs?NHCDs?with nitrogen-doped carbon nuclei were prepared by solvothermal method with citric acid and hydrazine hydrate as precursors and N-N-dimethylformamide?DMF?as solvent.Especially,comparing to non-doped hydrazide conjugated CDs,NHCDs exhibited 2.5-fold high ECL quantum efficiency because the lower potential could reduce notably the side reactions in the ECL process.Using the high-performance NHCDs to functionalize the electrode surface,a brief ECL biosensor was fabricated to detect hydrogen peroxide?H₂O₂?with the limit of detection as low as 7.6 nmol/L.Moreover,due to the high expression level of H₂O₂ in tumor cells than normal cells,the prepared ECL biosensor can quickly distinguish tumor cells from normal cells by detecting the expression level of H₂O₂ in 30min.2.Polyethylenimine-induced electrochemiluminescence enhancement of carbon dots and application to sensitive microRNA detectionIt is well-known that the co-reaction efficiency and the excited state quantum yield are the most important factors towards carbon dots?CDs?-based ECL efficiency.At present,the most common way to improve the ECL efficiency of CDs is to introduce co-reaction accelerators to increase the co-reaction efficiency and finally achieve the improvement of ECL efficiency.However,massive active intermediates produced in this way increase the risk of destroying biomolecules,so the increase of excited state quantum yield has more universal application value.In this work,polyethyleneimine?PEI?was used to wrap CDs to form PEI-CDs complex as an efficient ECL signal probe.Based on the PEI-CDs complex and synergistic hybridization-mediated chain replacement reaction,a sensitive ECL biosensor was constructed to detect microRNA,which showed wide linear range from 1 fmol/L to 1 nmol/L,and the limit of detection of 0.31fmol/L.Here,PEI not only enables CDs to achieve efficient enrichment,but also improves the ECL efficiency of CDs by 22.7%.The spectral characteristics of CDs indicated that the ECL emission of CDs originated from the localized excited state of the sp² carbon cluster coupled with the co-reaction site?hydrazide?.The interaction between PEI and the CDs increased the rigidity of the surface functional group and then increased the quantum yield of the localized excited state,which finally improved the ECL efficiency.This work described the characteristics of ECL localized excitation of CDs and defined the difference and relationship between photoluminescence and ECL.3.Bipedal DNA walker mediated enzyme-free exponential isothermal signal amplification for rapid detection of microRNAIsothermal enzyme-free DNA signal amplification technique,due to the advantages of wide applicability,low cost and simple operation,has been paid more and more attention in the field of bioanalysis,especially in the field of living cell analysis.The traditional isothermal enzyme-free DNA signal amplification technology is usually a linear signal amplification,and its signal amplification efficiency is not satisfactory.Therefore,in order to achieve efficient signal amplification,it is necessary to design an exponential signal amplification strategy.In view of the common problem of false positive in exponential signal amplification,a biped DNA walker driven by strand replacement reaction is used to mediate the release of secondary target,which achieves efficient isothermal enzyme-free exponential signal amplification.Based on the self-enhanced ruthenium?II?complex functionalized TiO₂ nanospheres as ECL signal probe,a highly sensitive ECL biosensor was constructed to realize the detection of microRNA in the range of 0.5 fmol/L¹0pmol/L,and the limit of detection was as low as 0.24 fmol/L.The isothermal enzyme-free exponential signal amplification strategy designed in this work provides a powerful tool for signal amplification in bioanalysis.4.Efficient electrochemiluminescence of carbon dots promoted by graphitic nitrogen doping and application to H₂O₂ analysisNitrogen atom doping in nitrogen-doped carbon materials has different structures such as pyridinic,pyrrolic and graphitic nitrogen,which have different effects on the properties of carbon materials.In the previous work,we have reported that nitrogen doping could reduce the anodic ECL potential of carbon dots?CDs?and improve the ECL efficiency.However,the specific doping structure is not clear.In this work,the effects of different nitrogen atoms doping on the oxidation potential of CDs were analyzed by the help of quantum chemistry.It indicated that graphitic nitrogen atoms were the key role to reduce the ECL excitation potential.Besides,we also have investigated the effect of graphitic nitrogen doping on the electrostatic potential near the hydrazide group and the results demonstrate that the distance from the graphitic carbon atom and hydrazide group is the key of the graphitic carbon atom to affect the reaction activity of the hydrazide group.Inspired by the results,graphitic nitrogen-doped CDs were first synthesized by solvothermal method using citric acid and urea as precursors,and then the surface functional groups of CDs were further hydrazinized to prepare a new type of anodic ECL rich graphitic nitrogen-doped CDs.Due to their higher graphitic nitrogen content,the prepared CDs have an ultra-low ECL excitation potential?0.45V?.In addition,semicarbazide-embedded CDs are synthesized by hydrothermal method using citric acid and semicarbazide as precursors,in which the graphitic nitrogen atom is adjacent to the hydrazide group,making the activity of the hydrazide group greatly improve.Interestingly,the ECL efficiency of the semicarbazide-embedded CDs is 35.1 times higher than that of randomly doped CDs and 73.8times higher than that of undoped CDs,respectively.Based on the prepared efficient ECL electrochemiluminescent CDs,a simple ECL assay has been constructed for H₂O₂ detection with a lower limit of detection?0.32 nmol/L?.This work illustrates the mechanism of nitrogen doping on the ECL excitation potential and anodic ECL efficiency of CDs from the specific structural level,which provides an important theoretical basis for the design and preparation of high performance ECL CDs.