Research On Detection Methods Of Fusarium Mycotoxins Based On Aptamer-functionalized Metal-organic Frameworks

Fusarium mycotoxins pose a significant risk to public health,agricultural production and international trade.However,the Fusarium mycotoxins detection techniques developed so far have struggled to meet the growing needs of consumers and regulators,and there is an urgent need to develop novel rapid sensing strategies for Fusarium mycotoxins.Metal-organic frameworks（MOFs）have unique physical and chemical properties and show promising applications in food safety detection.Nucleic acid aptamers are emerging bio-recognition elements that can be integrated into a myriad of distinctive DNA nanostructures to achieve customized functionality,and can further enhance the dispersibility,biocompatibility and sensing performance of MOFs-based bioprobes.However,MOFs-based aptasensors still face the drawbacks of low detection sensitivity and integration,poor ease of use and versatility,which pose significant barriers to the application of such sensors for the detection of food safety hazards.To overcome these shortcomings,a series of MOFs-based nanomaterials were synthesized,and the structural and functional properties of nucleic acid aptamers were comprehensively utilized,resulting in the construction of five stimuli-responsive MOFs-based aptasensors targeting Fusarium mycotoxins.The established optical sensing methods effectively enhanced the detection sensitivity of Fusarium mycotoxins,improved the ease of use and versatility of the strategies,shortened the detection time,and provided a new idea for the development and application of MOFs-based aptasensors.Firstly,a robust and enzyme-free"turn-on"type fluorescent aptasensor was developed for the sensitive identification of zearalenone（ZEN）in samples.On the one hand,the hydrothermally synthesized Fe-MOF was subjected to two-step calcination,and the obtained MOF-derived porous carbon was used as the energy receptor.On the other hand,a multifunctional ss DNA probe was designed.The C-rich template domain,located at the 5’end of the probe,was used to synthesize silver nanoclusters（Ag NCs）,the energy donor.Taking advantage of the conformational change of the probe induced by target binding,the G-rich enhancer domain at the 3’end of the probe can significantly increase the fluorescence intensity of Ag NCs through G-proximity enhancement effect.By combining the good magnetic and quenching ability of magnetic porous Fe₃O₄/carbon octahedra with the satisfactory photostability and water solubility of DNA-Ag NCs,the proposed FRET-based sensing strategy has excellent analytical performance.The linear range of detection was from 0.005 to 250ng/m L,and the LOD was 2×10^-3 ng/m L.Secondly,in order to obtain a more universal signal amplification element,a colorimetric aptasensor for the recognition of ZEN was developed based on stimuli-responsive aptamer-functionalized MOF-based nanocontainers and a trivalent G-quadruplex DNA mimicking enzyme（G4 DNAzyme）.Among them,the azide group-functionalized MOF was used as a nanocontainer to capture the catalytic cofactor,hemin.DNA hybrid duplexes were used as biological gates to assist in the completion of controlled release.In addition,a trivalent G4DNAzyme was used for signal amplification to improve detection sensitivity.Under optimal experimental conditions,the sensor had a linear range of 0.01 to 100 ng/m L with LOD as low as 0.36 pg/m L.It also exhibited excellent specificity,reproducibility,storage stability and reusability.Thirdly,in order to further improve the integration of MOFs-based aptasensor and shorten the detection time under the premise of ensuring the detection sensitivity,a colorimetric aptasensor with a refined design was developed based on MOFzyme and hyaluronic acid-DNA hydrogel.In this case,the hyaluronic acid-DNA hydrogel was deposited on the surface of the bimetallic MOFzyme via HCR.Dependent on the ZEN-specific aptamer integrated in the hydrogel network structure,the ZEN molecules can specifically trigger the disintegration of the hydrogel network and the concomitant exposure of the encapsulated MOFzyme.The degree of hydrogel disintegration was positively correlated with the amount of ZEN,thus allowing easy quantification of ZEN.The doping of the two M（IV）nodes resulted in a satisfactory oxidase-like activity of the Ce-Zr bimetallic MOFzyme,while effectively ensuring its stability.The DNA hydrogel ideally retained the recognition ability of the aptamer therein and was thus endowed with excellent stimuli-responsiveness.Due to the high integration of the sensor,the assay procedure was effectively simplified and the detection time was reduced to 45 min.Under optimal conditions,the sensor developed based on DNA hydrogel and MOFs-based catalytic nanomaterials had a linear range of 0.001-200 ng/m L and an LOD of 0.8 pg/m L.Fourth,based on the same principle,a colorimetric aptasensor was developed for the detection of fumonisin B₁（FB₁）by combining porphyrin-based MOF with polyacrylamide-DNA hydrogel,to further verify the versatility of the system developed based on DNA hydrogel and MOFs-based catalytic nanomaterials.First,porphyrin-based MOF with excellent peroxidase-like activity and moisture resistance was obtained by encapsulating hemin in the pores of MOF by an in situ synthesis method.Subsequently,polyacrylamide-DNA hydrogel was deposited on the surface of porphyrin-based MOF using chain-induced HCR.Likewise,the integrated aptamer sequences in its structure endowed the DNA hydrogel with stimuli-responsiveness to FB₁.After optimizing the experimental parameters,the sensor obtained a wide linear range from 0.05 to 100 ng/m L with LOD as low as 0.024 ng/m L.It also exhibited good specificity and storage stability.This study further provided strong evidence for the feasibility of colorimetric sensing strategy based on DNA hydrogel and MOFs-based catalytic nanomaterials.Fifth,in order to further improve the sensitivity of the system developed based on DNA hydrogel and MOFs-based catalytic nanomaterials,a colorimetric aptasensor for the quantification of FB₁ was developed based on DNA tetrahedra-functionalized magnetic beads and pure DNA hydrogel-coated Mn-Zr bimetallic MOFzyme.The DNA tetrahedra modified on the surface of the magnetic beads can effectively control the distribution density and orientation of the nucleic acid probes,thus improving the target capture efficiency.The doping of bimetallic nodes brought satisfactory catalytic capability to the Mn-Zr bimetallic MOFzyme.In response to a single target molecule,a DNA hydrogel network based on entropy-driven catalytic（EDC）reaction can be sequentially cleaved at both the main chains and cross-linking points,thus conferring extraordinary stimuli-responsiveness to this DNA hydrogel.Under optimized conditions,a linear range of 5×10^-4 to 50 ng/m L and an LOD of 0.38 pg/m L were obtained.Moreover,thanks to the superior versatility of this strategy,it can be easily extended to the detection of other food safety hazards by modifying very few bases in the DNA hydrogel network without optimizing the relevant experimental conditions.