Construction Of Aptamer Photoluminescent Nanoprobes And Their Applications For Cadmium (Ⅱ) Detection In Shellfish

Cadmium is a kind of toxic heavy metal that could be accumulated in the food chain and have adverse effects on food safety and consumer health.Humans might consume food that contained excessive cadmium through diet.The overload cadmium could be accumulated in the human body and become a long-term threat to human health.Once the foods with excessive cadmium being consumed by humans,the overload cadmium could be accumulated in the body and become a long-term threat to human health.In recent years,people have become more concerned about seafood quality,especially the heavy metal content level in shellfish.Shellfish,as the filter feeders,are easily absorbed and accumulated with cadmium in water or sediment.The shellfish have a long tolerance range to heavy metal,which means the cadmium is easily accumulated in shellfish.Therefore,it is necessary to detect and control cadmium content in shellfish products in production,processing,packaging,and transportation.The traditional laboratory cadmium detection methods require expensive experiment equipments and require a trained laboratory technician with certain professional skills.Also,tedious and time-consuming pre-processing procedures are needed.The lack of maneuverability,economy,and portable has become the drawback of these detection methods,limiting the application of these methods in rapid testing and field testing,especially for the routine cadmium detection jobs.In this work,aptamers were used as molecular recognition tools,combined with the fluorescent carbon nanoparticles from food and long afterglow phosphorescence nanoparticles as photoluminescence markers to build a series of rapid,simple and accurate detection methods for the analysis of cadmium in food samples.Firstly,the food origin fluorescence carbon nanoparticles（FCNs）were found in both roasted and water boiled sweet potatoes.Compared with the chemical synthesis of FCNs,the FCNs collected from food processing is economical,environmentally friendly and simple instructions.The corresponding average size of the FCNs from sweet potato peel after 250 ℃ roasting for 20,40,and 60 minutes was approximately 7.0,2.8,2.4 nm,respectively.And the average size of FCNs from 60 minutes of roasted sweet potato pulp was about 2.1 nm.Moreover,FCNs’average size from peel and pulp of sweet potato after boiling at 100 ℃ for 60 min was about 6.0 and 1.7 nm,respectively.Besides,the peak of the X-ray Diffraction（XRD）spectrum of FCNs could be attributed to highly disordered carbon atoms.Fourier Transform Infrared（FT-IR）spectroscopy was employed to identify the surface of FCNs contains multiple functional groups.And the elemental composition was further characterized by X-ray Photoelectron Spectroscopy（XPS）spectroscopy,and the peaks could be assigned to C,N,and O,respectively.The calculated quantum yield of FCNs in the 60 minutes roasted sweet potato peel was 2.3%,which were also provided good p H stability and be chosen as the fluorescent markers for further use.Secondly,the persistent luminescence nanorods doped with divalent manganese ions were synthesized.The signal of persistent luminescence nanorods is not easily interfered with by the fluorescence of the detection sample matrix.The reaction time and reaction temperature were chosen as variables in the hydrothermal synthesis.The Zn₂Ge O₄:Mn nanorods had rod-shaped morphology with clear lattice fringes.With temperature increased from 200 to 240 ℃,the average length of the nanorods grew from about 45 to 80 nm,and the average width slightly increased from about 17 to 28 nm.Along with increasing reaction times（2,4,6,8 h）at 200°C,the average length of nanorods increased slightly from about 45 to 65 nm,and the average width increased gradually from about 17 to 25 nm.Moreover,the nanorods’elemental composition was characterized by XPS spectroscopy,and the peaks indicated the binding energies of Zn,Ge,Mn,and O elements.The Zn₂Ge O₄:Mn nanorods showed visible green luminescence under the illumination of a 254 nm UV lamp,with the maximum emission peaks at 540 nm.The phosphorescence lifetimes of the Zn₂Ge O₄:Mn nanorods were between 16.32 ms and 17.33 ms,which displayed long-lifetime luminescence.The Zn₂Ge O₄:Mn nanorods were chosen as the phosphorescence markers due to their long afterglow property.Thirdly,the application of FCNs extracted from roasted sweet potato peel was designed as energy donors to construct a method for detecting cadmium in food samples based on the fluorescence resonance energy transfer study.FCNs were coupled with dopamine and then linked with aptamers which were used as fluorescent probes with recognition ability in this study.The magnetic reduction graphene oxides were used as energy accepter in the Fluorescence Resonance Energy Transfer（FRET）system.When the cadmium ion is absent,the fluorescent probe is adsorbed to the surface of the magnetic graphene oxide,and the fluorescence of the fluorescent carbon nanoparticles is quenched by FRET;and when the cadmium ion is present,the aptamer departs from the magnetic oxidation Graphene surface due to a stronger affinity with the cadmium ion and inhibits FRET.Under the optimized reaction conditions,the concentration of cadmium ions could be detected with this method.Within the range of 1-55 ng·m L^-1,there is a linear relationship between cadmium ion and fluorescence signal recovery,and the lowest detection limit is 0.1 ng·m L^-1.The result confirmed that this method could be used for the detection of cadmium ions in real samples.Fourth,the application of long afterglow nanorods as phosphorescent energy donors in Phosphorescence Resonance Energy Transfer（PRET）is studied to reduce interference by the fluorescence of the sample matrix.The long afterglow nanorods were used as phosphorescence energy donors in phosphorescence resonance energy transfer is studied in this method.The cadmium aptamers were connected with magnetic nanoparticles,which were formed as the magnetic recognition probe.Then the quenching group modified the complementary chains of aptamer（Black Hole Quenchant 1-c NDAs,BHQ₁-c NDAs）were hybridized with the probe.The long afterglow nanorods were used as the signal element in this method.In this detection mode,the cadmium ions could be binding to the cadmium aptamer and be removed through magnetic separation.The BHQ₁-c NDAs were released from aptamer and quenching the phosphorescence signal by phosphorescence resonance energy transfer.The phosphorescence of the signal element is quenched by PRET,which build the"on-off"type of cadmium ion detection.A sensitive aptamer sensor to detect cadmium in food has been successfully established,and the lowest detection limit is 0.04 ng·m L^-1.This experiment lays a foundation for the detection of cadmium ions in long afterglow time-resolved mode.Fifthly,on the basis of the previous method,we have explored the mode of using long afterglow nanorods as energy donors to establish a signal-enhanced detection method for PRET.Compared with the"on-off"phosphorescence quenching sensor,the"off-on"phosphorescence signal enhancement sensor can reduce the false positive interference of photobleaching and sensitive detection environment on the detection result.A signal enhanced cadmium detection method is established designed through a phosphorescence resonance energy transfer system.The long afterglow nanorods were used as the energy donor and connected with aptamer to form the recognition probe.The black hole quenchant 1（BHQ₁）is used as the energy acceptor modified with the aptamer’s complementary chain.The aptamer and the complementary chain bonded together,and BHQ₁ could quench the phosphorescence signal.With the appearance of cadmium ions,the cadmium ions could preferentially combine with the aptamers,and the PRET system would be broken and restore the phosphorescence signal.There is a good linear relationship under optimal detection conditions in the range of 0.5-50 ng·m L^-1,and the lowest detection limit is 0.35 ng·m L^-1.The results show that this method has demonstrated good specificity and high sensitivity and improved cadmium detection performance in food samples.In conclusion,the food origin fluorescence carbon nanoparticles and long afterglow nanorods were used as the photoluminescence detection elements.Combining with the aptamers,a series of simple,sensitive and easy-to-use detection methods for analysis cadmium in shellfish were constructed in this paper.Moreover,these established detection methods provide the support of expanding the usage of aptamer and being the technical support for the food safety detection system.