Fluorescence Sensing Strategies Based On Functional Nucleic Acidinduced Isothermal Enzyme-free Amplification For Detection Of Heavy Metals And Antibiotics

Heavy metals and antibiotics are typical organic and inorganic pollutants in food and the environment.Heavy metals are biologically toxic,carcinogenic,mutagenic,and produce allergic reactions.Antibiotics can induce bacterial resistance,human flora imbalance,drug allergic reactions,and organ damage.Both the accumulation of heavy metals and the overuse of antibiotics pose serious threats to human health and ecosystems.Therefore,the analysis of heavy metals and antibiotics in food is an effective technical means to monitor and ensure food quality and safety.Many countries have established maximum residue limits in food and the environment to protect people from these chemical contaminants.At present,the detection of heavy metals and antibiotics is mainly carried out by large-scale instrument analysis methods.The detection of heavy metals often adopts analytical techniques such as atomic absorption spectroscopy,atomic fluorescence spectrometry,and inductively coupled plasma mass spectrometry.The detection of antibiotic residues mostly uses high performance liquid chromatography,gas chromatography,chromatography-mass spectrometry and other analytical techniques.These methods are well-developed and have the advantages of high sensitivity and good reproducibility.However,due to the complex pretreatment,the bulky and expensive instruments,the large and time-consuming detection reagents,and the need for professional and technical personnel to operate,these methods limit to a certain extent.its practical application.Therefore,the development of a simple,rapid and reliable technology to detect chemical hazards is of great significance to environmental protection,food safety and human health.In recent years,with the development of biotechnology,the special biological functions and flexible spatial structure of functional nucleic acids have effectively improved the specificity of detection,and have been used to construct various biomolecular recognition probes with different spatial conformations.The application of biosensing and analysis technology in food and environmental samples is more and more widely in the field of chemical contaminant detection.At the same time,nucleic acid isothermal amplification technology,as a new nucleic acid in vitro amplification technology,can achieve rapid and highly sensitive amplification of target molecules under constant temperature conditions.This technology has the advantages of convenient operation and does not need to rely on precise temperature-controlled instruments and equipment.It has been widely used in the detection of various biomolecular target substances and plays an important role in many research fields.Combining the actual situation of heavy metal and antibiotics residues in food and the environment,this paper aims at four typical target molecules,based on functional nucleic acid and nucleic acid enzyme-free isothermal amplification technology,using fluorescent dyes and silver nanoclusters with excellent optical properties as the output Signal,four fluorescent"signal on"biosensors with simple operation,low cost and good sensitivity were constructed to detect and analyze residual heavy metals and antibiotics in the environment and food.The monitoring platform provides reference.The main research contents include:(1)A label-free and enzyme-free signal-on biosensor for Hg²⁺was constructed based on T-Hg²⁺-T nanoladders and fluorescent DNA-templated silver nanoclusters/graphene oxide nanocomposites(DNA-Ag NCs/GO).The T-rich sequences(P1 and P2)were used to capture Hg²⁺and form the T-Hg²⁺-T nanoladders.C₆G₅C₆ was extended at the 5'end of P1,used as a template for synthesizing fluorescent DNA-Ag NCs.In the presence of mercury ions,the T-Hg²⁺-T nanoladders caused significantly enhances the emission of Ag NCs.GO can attract free P1-C₆G₅C₆ and P2that are not involved in the formation of T-Hg²⁺-T nanoladders,thus employed as the energy acceptor to quench the background fluorescence of Ag NCs.The fluorescence enhancement induced by mercury ions can be used for quantitative detection of Hg²⁺over the range 0.1?30 n M with a detection limit as low as 7.35 p M.This method was used to detect Hg²⁺in the spiked samples of Songhua River water and grass carp,with the recoveries of 97.61%?103.79%and 96.52%?105.58%respectively,consistent with the results from atomic fluorescence spectrometry.Obviously,in this assay,enzyme-free signal amplification and signal identification are integrated in the ingenious design of nucleic acid sequences,thus achieving the label-free fluorescence detection of mercury ions.(2)A fluorescence''signal on''strategy for lead ions detection was established in combination of Pb²⁺-specific 8-17 DNAzyme with catalytic hairpin assembly(CHA).8-17 DNAzyme was used as the recognition element of Pb²⁺,which catalyzed the cleavage of an RNA base embedded in the DNA substrate in the presence of Pb²⁺,while releasing part of the substrate strand(S')as CHA initiator.And two hairpin probes(H1and H2-FQ)were designed according to the sequence of S'for CHA,in which H2-FQ was labeled with fluorophore FAM and quencher BHQ-1 as fluorescent"molecular switch"based on fluorescence resonance energy transfer(FRET).S'can hybridize with the ring of H1 to open the hairpin structure of H1,and expose the stem sequence of H1to the solution for further opening hairpin H2-FQ,moving FAM away from BHQ-1 and reducing the efficiency of FRET between them,thereby realizing the"signal on"fluorescence strategy.At the same time,S'was displaced from H1 due to the double-stranded H1/H2-FQ is more stable compared to S'/H1 complex.The displaced S'can initiate a new cycle of CHA,forming a large amount of H1-H2 complexes,and the enzyme-free isothermal amplification was achieved.With the linear range of 0.5?1000n M and the detection limit of 0.5 n M,this amplification method has better detection performance compared to the FQ-labeled 8-17 DNAzyme method.The established biosensor exhibits good specificity and can be effectively used for the detection of Pb²⁺in real spiked samples of river water and grass crap.DNAzyme and CHA are integrated in the ingenious design of nucleic acid sequences to realize the enzyme-free isothermal amplifications and sensitive detection of Pb²⁺,which holds potential versatility in food supervision and environmental monitoring.(3)An enzyme-free amplification fluorescent strategy was developed for sensitive detection of kanamycin based on three-way toehold-mediated strand displacement(TMSD)via Lr RET between FAM and graphene oxide(GO).Three metastable hairpin DNA probes with fluorophore were designed as the assembly components to construct the sensing system.The toehold-mediated strand displacement of hairpins was induced with the help of helper DNA(HD)by the specific binding of the aptamer to kanamycin.Enzyme-free signal amplification was obtained through TMSD reactions to open the hairpins and recycle the HD.The formed rigid DNA triangles containing double helix of DNA(ds DNA)was stiffer,and the sticky end of the unreacted hairpins will be closely adsorbed onto GO surface via?-?stacking to quench the fluorescent signal.The"off-on"type fluorescence signal was generated to achieve quantitative detection of kanamycin.This strategy has good sensitivity and selectivity for kanamycin with a detection limit of 1 n M,and has been successfully applied to the detection of spiked milk samples.The proposed biosensor is simple and convenient in operation,requiring only the mixing of several solutions at room temperature to directly detect without complex instrument,and provides a new approach for the monitoring of kanamycin in food safety analysis.(4)Based on hybridization chain reaction(HCR)and fluorescence synergism,a novel aptasensor for tobramycin was successfully fabricated.Tobramycin competed with c DNA-FAM to bind aptamers immobilized on magnetic beads.After magnetic separation,the released c DNA-FAM acted as initiator to trigger HCR amplification,thus the fluorescence was significantly enhanced due to binding of SYBR Green?(SG?)to the formed long double-stranded DNA and the synergistic fluorescence of FAM.In the absence of tobramycin,the initiator was magnetically separated and no HCR occurred,more importantly,graphene oxide can quench the fluorescence of excessive hairpins/SGI and c DNA-FAM,so almost no background signal was detected.This aptasensor can monitor tobramycin in the range of 0.3?50?M with low detection limit of 17.37 n M.Due to the potential generality of structure-switching aptamers and effectiveness of fluorescence synergism,this enzyme-free amplification strategy can be extended to other applications by rational design of nucleic acid sequences.