Terbium(Ⅲ)-Referenced N-doped Carbon Dots For Ratiometric Fluorescent Sensing Of Mercury(Ⅱ) In Seafood

With the improvement of people’s living standard,food safety has become a major global concern.Food safety incidents caused by seafood not only seriously endanger human health,but also may become the reason for food trade disputes between countries.As a virulent heavy metal,mercury（Hg）can be enriched in marine animals and plants through human and natural activities.Long-term consumption of seafood contaminated by Hg²⁺can lead to headaches,dizziness,muscle tremors,motor and cognitive dysfunction and cardiovascular diseases.Conventional analytical methods for detecting Hg²⁺are limited by rigorous analytical procedures,high cost and insufficient selectivity in complex food matrix.Therefore,rapid and sensitive monitoring Hg²⁺residues in seafood can protect the rights and interests of food safety of human and promote the healthy development of seafood trade.Nanomaterials-based fluorescent sensing holds great potential in designing highly sensitive and selective detection strategies for food safety detection.As a new organic quantum dots,carbon dots（CDs）present the advantages of photochemical stability,ready solubility,high biocompatibility and no/low-toxicity.Moreover,Heteroatom doping is an efficient approach to provide abundant active groups on the surface of CDs and improve fluorescence properties of CDs.It can also enhance the specificity of its target recognition.In order to overcome the challenge of lower sensitivity and selectivity of the single-emitting CDs sensors interfered by intricate food matrices.Nanohybrid CDs based ratiometric fluorescent sensing with self-calibration is regarded as an efficient strategy for the specific recognition of target analyte in the food matrices.In this study,a ratiometric fluorescence（FL）sensor was fabricated by coordinating 2,6-pyridinedicarboxylic acid（DPA）sensitized Tb³⁺（Tb-DPA）with-NH₂ and-COOH on the surface of the N-doped carbon dots（N-CDs）directly at room temperature for detecting Hg²⁺in seafood.The main research results are as follows:1、N-CDs are synthesized by the hydrothermal method using 4.2 g citric acid and6.0 g urea as precursors and heated to 180°C for 4 h.The morphology and fluorescence quantum yield of N-CDs are characterized.TEM reveals that the obtained N-CDs are spherical and uniformly dispersed and the particle size of N-CDs was 0.84-7.50 nm with an average diameter of 4.48 nm.N-CDs had a higher quantum yield（QY）of 32.74%than that of 18.34%of CDs because N-doping introduced a new surface state labeled as the N-state,it significantly enhances the fluorescence intensity of N-CDs.10 m L DPA aqueous solution（10 m M）was mixed with 0.02 g Tb Cl₃·6H₂O to form a clear solution.Then the solution was added into 54 m L N-CDs（1 mg/m L）and was stirred overnight.The optical,stability and surface functional groups of the obtained N-CDs-Tb-DPA are characterized.FTIR demonstrate that O atom of-COOH and N atom of-NH₂ of N-CDs can directly coordinate with Tb atom in Tb-DPA to form the N-CDs-Tb-DPA sensor.The N-CDs-Tb-DPA sensor exhibits two characteristic emissions at 436 nm of N-CDs as the response signal and at 543 nm of Tb-DPA as the reference signal when excited at 290 nm for detecting Hg²⁺in seafood.The fluorescence intensity of the two emission peaks of the sensor(F₄₃₆ and F₅₄₃)show good photostability when stored at room temperature for one month（P>0.05）.FTIR and XPS analyses demonstrate that N-CDs-Tb-DPA is successfully synthesized with the abundant active groups of-OH,-COOH and-NH₂,providing potential recognition sites for chelating Hg²⁺.2、The recognition mechanism of N-CDs-Tb-DPA sensor to Hg²⁺was evaluated by XPS spectrum.Hg4f could be de-convoluted into three peaks at Hg,Hg-O(Hg4f_7/2)and Hg-O(Hg 4f_5/2)demonstrate that Hg²⁺could be chelated by-COO^-on the surface of N-CDs,and binding to the sensor is mainly in oxidation state.The higher capability of electron donation of Hg²⁺than-NH₂inhibited ET between-NH₂ and-COOH,enhancing binding energy of N-H from 401.69 e V to 401.85 e V,weaking the electron shielding effect of N-H,quickly quenching the FL of N-CDs by changing the electron density of N.And the effect of Hg²⁺on the fluorescence intensity of Tb-DPA was negligible compared with that of N-CDs.Under the optimal conditions,that is,the detection system p H is 7.0,the incubation time is 420 s and the chosen incubation temperature is 25°C,a good linear relationship between the fluorescence intensity ratio of N-CDs-Tb-DPA+Hg²⁺(F₄₃₆/F₅₄₃)and Hg²⁺concentrations of 1-161.51μM is obtained.The calibration equation was described as F₄₃₆/F₅₄₃=-0.043X+6.959with the coefficient of determination（R²）of 0.996(X is the concentration of Hg²⁺,μM).The limit of detection（LOD）is calculated to be～37 n M.3、Common interfering ions in seafood(Zn²⁺,Al³⁺,Mn²⁺,Pb²⁺,Cr³⁺,Ag⁺and Cu²⁺with 3-fold concentration of 36.49μmol/L Hg²⁺and Na⁺,K⁺,Mg²⁺,Ca²⁺and I^-with 10-fold concentration of 36.49μmol/L Hg²⁺)have no significant effect on the Hg²⁺detection results（P>0.05）,demonstrating that the sensor possessed outstanding selectivity and anti-interference ability for the determination of Hg²⁺.The developed N-CDs-Tb-DPA sensor was used to carry out standard addition recovery tests on shrimp,kelp,octopus and large yellow croaker samples.The contents of Hg²⁺in four seafood samples of fish,shrimp,sleeve-fish and kelp are determined by the developed N-CDs-Tb-DPA sensor.The recoveries of 86.45-114.47%with low relative standard deviations（RSD,n=3）of 0.20-1.92%are achieved,demonstrating that the sensor for Hg²⁺detection in real seafood samples is highly reliable and feasible.In this study,a sensitive,convenient,low-cost and anti-interference ratiometric fluorescence sensor is constructed to detect Hg²⁺in seafood,which paves a new avenue for the design of novel ratiometric FL sensors for potential applications in food safety.