Synthesis Of Carbon Dots And Their Applications In Biosensing And Pollutant Detection

Carbon dots（CDs）,a novel carbon-based nanomaterial with a wide range of applications,were first discovered by chance in 2004 by Xiaoyou Xu et al.Compared to conventional semiconductor quantum dots and organic small molecules,CDs are widely used to construct nanofluorescence probes due to their simple synthesis methods and excellent physico-chemical properties（low toxicity,good water solubility,biocompatibility,fluorescence tunability,and abundant surface functionalities,etc.）,and have promising applications in environmental detection,bioimaging,drug loading,and traceability.Therefore,it is of great significance to design and synthesize novel fluorescence probes for the target,improve the sensitivity and selectivity of detection,and expand their application in the field of analysis.This dissertation consists of five chapters and four research works.The first chapter introduces the research background of this paper.We briefly describe the definition,properties,synthesis methods,detection mechanism of probes and the latest progress in the application field of CDs.In the subsequent chapters,we used citric acid,chitosan,neutral red and other materials as precursors to prepare four different functionalized CDs.Based on fully characterizing the properties of CDs,we established enhanced,quenched and ratiometric fluorescence sensing systems based on the prepared CDs,and applied them to biological samples（cysteine and alkaline in cells）.Phosphatase),environmental samples（malachite green）,and detection of water in organic solvents.This dissertation is devoted to optimizing the synthesis methods of CDs,developing fast and efficient methods for the preparation of CDs and enriching the application of carbon dots in biological and environmental detection.The specific research work is as follows:（1）Independently excited fluorescent CDs were synthesized by a one-step hydrothermal method using citric acid and ethylenediamine as raw materials,and their structures and properties were studied in detail.Since the excitation spectrum of CDs overlaps with the absorption spectrum of Ag NPs,resulting in the inner filter effect（IFE）,which effectively suppresses the fluorescence emission of CDs,the fluorescence intensity of CDs decreases.However,when the analyte cysteamine was added,a stronger interaction was formed between the-SH of cysteamine and Ag NPs,resulting in the recovery of the fluorescence of CDs.Based on this,a fluorescent"off-on"probe was constructed and successfully applied to the quantitative detection of cysteamine.Cysteine showed a good linear correlation（limit of detection is 0.35μM）with the fluorescence intensity of CDs in the concentration range of 2-16μM,and the correlation coefficient r was 0.9979.The CDs have the characteristics of simple synthesis,high sensitivity,and good cytocompatibility.They have been expanded to detect cysteamine in bovine serum,and show good stability and reproducibility.Furthermore,based on the low cytotoxicity of the CDs,we applied this method to the imaging experiments of Hep G2 cells and explored the feasibility of intracellular cysteamine detection.The results confirmed that the constructed fluorescent probe can be used for the detection of cysteamine in vitro and in vivo,and the fluorescent probe has the characteristics of mild detection conditions,low detection limit and high specificity,and has good performance in the fields of drug treatment and early diagnosis of diseases.application prospects.（2）Hydrophilic C-CDs were prepared by a one-step hydrothermal method using chitosan and ethylenediamine as raw materials.A ratiometric fluorescent probe based on C-CDs and calcein was constructed,and the sensitive detection and cell imaging analysis of alkaline phosphatase（ALP）were realized by utilizing the inner filter effect（IFE）mechanism.First,the optimal emission peaks of C-CDs and calcein are located at 412 nm and 512 nm,respectively.Since the UV absorption peak of calcein overlaps with the optimal emission peak of C-CDs,based on the inner filter effect,the fluorescence emission peak of C-CDs is quenched.After adding Eu³⁺to the system,Eu³⁺was able to combine with calcein and cause the fluorescence of calcein at 512 nm to be significantly quenched.Secondly,the phosphate ion(PO₄^3-)generated by the hydrolysis of its substrate p-nitrophenylphosphate disodium salt（p NPP）by ALP has a higher affinity for Eu³⁺than calcein,resulting in the quenching effect of Eu³⁺on calcein fluorescence attenuated,the fluorescence of calcein at 512 nm was restored.At this time,the fluorescence of C-CDs was quenched again due to the IFE.By measuring the ratio of fluorescence intensity of C-CDs and calcein in the system,a highly sensitive and selective detection of ALP concentration was achieved.The linear range of the fluorescent probe system for the determination of ALP was 0.09-0.8 m U m L^-1,and the detection limit was as low as 0.013 m U m L^-1.The method was validated with human serum spiked samples,and good spiked recovery rate was obtained.In addition,this new assay can be applied to screen for ALP inhibitors as well as to detect ALP in human serum.Further cell imaging experiments showed that the method showed good biocompatibility in Hep G2 cells,and was suitable for intracellular monitoring and imaging of endogenous ALP,which has the prospect of biomedical research and disease diagnosis.（3）A one-step method was used to synthesize BPEI-NS and Mn O₂ NS at lower temperature,and a facile fluorescence system was developed to realize the sensitive quantitative analysis of MG in fish samples.The detection mechanism is as follows:Mn O₂ NS were used as energy acceptors to quench the fluorescence of BPEI-CDs at445 nm by fluorescence resonance energy transfer（FRET）.When butyrylcholinesterase（BCh E）coexists with the substrate S-butyrylthiocholine iodide（IBTCh）,BCh E catalyzes IBTCh to generate thiocholine（TCh）,which can effectively decompose Mn O₂ NS to Mn²⁺,thereby restoring the fluorescence of BPEI-CDs.As an inhibitor of BCh E enzymatic activity,the presence of MG can inhibit the production of thiocholine,which in turn inhibits the decomposition of Mn O₂ NS,resulting in the inhibition of the fluorescence of BPEI-CDs in the system.Based on this,we established a quenched fluorescence system,using the fluorescence intensity at 445 nm as an indicator signal to achieve quantitative detection of MG concentration,and proved that this method has excellent performance by measuring MG concentration in spiked fish samples.The analysis tests performance and usability.（4）Preparation of red light-emitting TN-CDs using neutral red and tartaric acid using a one-pot method.At an excitation wavelength of 520 nm,TN-CDs emit red fluorescence and exhibit interesting solvent-dependent effects.When trace amounts of water are present in organic solvents such as ACN,Et OH,Me OH,and DMF,the fluorescence of TN-CDs in solvents gradually decreases.In the DMF solution,the color of the mixture can be observed with the naked eye,and it is found that with the increase of water content,it changes from yellow to pink to purple.Furthermore,to develop a fast,convenient,micro-detection method and obtain accurate concentration results,We combine paper strips,UV light sources,and smartphones for image acquisition,color output,and data fitting.The fluorescence intensity of TN-CDs was linearly correlated with the water content in DMF at 0-100%water content,the correlation coefficient r was as high as 0.9983,and the RSD value of the spiked samples was≤3.5%.