Novel Composite Fluorescent Materials And Biosensing Strategies Based On Carbon Dots And Gold Nanoclusters

Sensitive detections of pharmaceuticals,metabolites,enzymes and energy-carrying molecules in organism are crucial for biochemical analysis and clinical assessment,diagnosis,or treatment.Fluorescence spectrometry is widely used in the field of biochemical analysis due to its high sensitivity,rapid response and simple operation.Preparation and selection of fluorescent nanomaterials are the key points of the researches in constructing fluorescence sensing strategies.Carbon dots（CDs）,as a kind of zero-dimensional carbon nanomaterials,possess good water dispersibility,low toxicity,good biocompatibility,tunable fluorescence emission,and strong photobleaching resistance.Gold nanoclusters（Au NCs）consisting of several to hundreds of gold atoms exhibit the properties of large Stokes shift,good photostability and easy chemical modification.The combination of CDs and Au NCs will facilitate the development of fluorescence sensing strategies with superior analytical performance.In this work,four kinds of CDs with different luminescence properties were prepared by hydrothermal method using citric acid,ethylenediamine,diethylenetriamine,ethylenediamine tetraacetic acid,and calcein as precursors,and Au NCs were synthesized using glutathione as template.Then,four novel ratiometric fluorescence sensing strategies for detecting pharmaceutical and biomolecules in human fluids were constructed based on the prepared CDs and Au NCs.Mechanisms and performance of these strategies are as follows:（1）Blue-emission CDs were prepared by a facile one-step hydrothermal method using citric acid and ethylenediamine as reactants.A sensing strategy employing both ratiometric fluorometry and colorimetry for detecting copper ions(Cu²⁺)and D-penicillamine（D-pen）was developed by utilizing o-Phenylenediamine（OPD）as a chromogenic development reagent.The oxidation reaction of OPD induced by Cu²⁺generates the oxidation product 2,3-diaminophenazine（ox OPD）,which not only emits green fluorescence at 555 nm,but also exhibits obvious absorption at 420 nm.Additionally,the blue fluorescence of CDs at 443 nm was quenched by ox OPD via the inner filter effect（IFE）and F?rster resonance energy transfer（FRET）of ox OPD on CDs due to both the spectral overlap and the suitable distance between CDs and ox OPD.The intense chelation affinity of D-pen to Cu²⁺resulted in the significant decrease of Cu²⁺amount in the testing system,that would inhibit the oxidation of OPD,and consequently change the fluorescence emissions of both ox OPD and CDs as well as the absorption of ox OPD.Therefore,the intensity ratio of fluorescence emitted from CDs to that from ox OPD(F₄₄₃/F₅₅₅)and the absorbance of ox OPD(A₄₂₀)were measured and calculated as the spectral response signals for sensing D-pen.This dual-signal sensing strategy was applied to selectively and sensitively detection of both Cu²⁺and D-pen.The limits of detection（LODs）of Cu²⁺and D-pen were 0.019μM and 0.092μM for the testing mode of ratiometric fluorometry,respectively.Also,the low cytotoxicity of the reagents involved in this sensing strategy facilitates its application in fluorescence imaging of live cells（2）Iron and nitrogen co-doped carbon dots（Fe/N-CDs）were prepared by a hydrothermal method using（ethylenedintrilo）tetraacetic,ferric chloride,and diethylenetriamine as the sources of carbon,iron and niteogen.A universal sensing strategy for detecting hydrogen peroxide（H₂O₂）and its related metabolites in human fluid was developed based on ratiometric fluorometry and colorimetry by utilizing the peroxidase-like property and the fluorescence emission at 449 nm(F₄₄₉)of the Fe/N-CDs.The oxidation of OPD by H₂O₂catalyzed by Fe/N-CDs generates ox OPD effectively,which can induce the F?rster resonance energy transfer（FRET）from Fe/N-CDs,which exhibits obvious absorption at 420 nm(A₄₂₀)and fluorescence emission at555 nm(F₅₅₅).F?rster resonance energy transfer（FRET）from Fe/N-CDs to ox OPD results in the quenched and enhanced fluorescence of Fe/N-CDs and ox OPD,respectively.The opposite variation trends of these fluorescence emissions facilitates the quantitation of H₂O₂by ratiometric fluorometry using a fluorescence intensity ratio(F₅₅₅/F₄₄₉)as the response signal.Similarly,absorbance of ox OPD can indicate the amount of H₂O₂ consumed during the oxidation reaction.By employing the corresponding oxidases,many metabolites such as xanthine and uric acid can be oxidized with the production of H₂O₂.Hence,this dual-mode sensing strategy is also suitable for sensitively and selectively monitoring metabolites involved in H₂O₂metabolism in human fluids.The LODs of H₂O₂,xanthine and uric acid obtained by this sensing strategy were 0.07,0.15,and 0.14μM for ratiometric fluorescence mode,and 0.12,0.52,and 0.47μM for colorimetric mode,respectively.This sensing strategy demonstrates good versatility for detecting other substances related to H₂O₂ metabolism in human fluids.（3）Blue-emission CDs were prepared by a one-step hydrothermal method using citric acid,ethylenediamine and calcein as reagents,and glutathione-capped Au NCs（GSH-Au NCs）were prepared using GSH as both reducing agent and template.A novel fluorescence sensing strategy for detectingα-glucosidase in human serum was proposed based on CDs and GSH-Au NCs.During theα-glucosidase catalyzed hydrolysis reaction of L-ascorbic acid-2-O-α-D-glucopyranosyl（AAG）,ascorbic acid（AA）was generated as a product,which would reduce cerium ion(Ce⁴⁺)to a lower valence state(Ce³⁺).Since Ce³⁺can selectively improve the aggregation of GSH-Au NCs,the fluorescence enhancement of GSH-Au NCs arised from aggregation-induced emission enhancement（AIEE）of is suitable for indicating the activity ofα-glucosidase.Additionally,the red fluorescence emission（650 nm）of GSH-Au NCs facilitates the elimination of autofluorescence emitted from protein.To eliminate the influence of fluctuation of instruments and environmental parameters,the fluorescence emission of CDs that is independent of the hydrolysis reaction,was also monitored as the fluorescence reference to realize ratiometric fluorometry.The linear response range toα-glucosidase activity and the LODs obtained by the proposed strategy were 0.01 U m L^-1-0.1 U m L^-1 and 0.0055 U m L^-1.Good consistency between results of serum sample examinations by using this strategy and commercial kits as well as the capacity of this strategy for live-cell imaging reveal the promising potential of this strategy for clinical diagnosis.The mothod for regulating aggregation state of GSH-Au NCs as well as the specific mechanism of fluorescence signal response applied in this sensing strategy have instructive significance for the application of similar fluorescent nanomaterials.（4）Blue-emission CDs were synthesized by a one-step hydrothermal method using citric acid and diethylenetriamine as carbon and nitrogen sources,respectively.GSH-Au NCs were prepared using the same method and reagents as the above work.A novel sensing strategy was developed for sensitive detecting adenosine triphosphate（ATP）based on the formation and degradation of zeolitic imidazolate framework-8（ZIF-8）structure encapsulating CDs and GSH-Au NCs（CDs/Au NCs@ZIF-8）.Under excitation at 360 nm,this nanocomposite exhibits dual-emissions at 469 nm and 660 nm,respectively,arising from CDs and the AIEE of Au NCs.ATP can selectively degrade the ZIF-8 framework in this nanocomposite via the coordination competition towards Zn²⁺ions,that are originally coordinated by 2-Methylimidazole to form the ZIF-8framework,resulting in the release and dispersion of CDs and Au NCs.The dispersion of released CDs results in the enhanced fluorescence emission due to the improvement of excitation efficiency.On the contrary,the dispersion of unrestricted Au NCs inhibits the AIEE and leads to decreased fluorescence emission of Au NCs.So the fluorescence intensity ratio(F₄₆₉/F₆₆₀)was conveniently employed as the response signal for indicating the amount of ATP consumed during the above competitive reaction.The LOD for detecting ATP by this strategy is 0.061μM.Besides,low cytotoxicity of this nanocomposite facilitates its application in fluorescence cellular imaging.The encapsulation,release and dispersion methods of aggregated CDs and GSH-Au NCs applied in this sensing strategy,as well as the corresponding fluorescence signal response mechanism,will promote the practical applications of the fluorescent nanomaterials with aggregation state-dependent emission.