Construction Of A Switch Fluorescence Biosensor Based On Zirconium Porphyrin MOF And Its Application In Bioassay

Objective:Based on the zirconium porphyrin metal-organic framework（MOF）and the fluorescent dye-labelled single-stranded DNA（ss DNA）,we constructed a simple,fast,ultra-sensitive and highly specific switch fluorescent biosensing method for the detection of nucleic acid biomarkers,which could provide the technical guarantee for personal health and public safety.Methods:A crystalline porous materials of zirconium porphyrin metal-organic framework（MOF）,namely ZrMOF,was synthesized by the hydrothermal method.We used ZrMOF as a high efficiency fluorescence quencher and 6-carboxyfluorescein dye（FAM）-modified ss DNA sequence（FAM-P1/P2）as a specific recognition element.ZrMOF could effectively adsorb FAM-P1/P2 and strongly quench the fluorescence of FAM.In the presence of target analytes,the FAM-P1/P2 bound specifically to them and were released from the ZrMOF surface,inducing the fluorescence recovery of FAM.Accordingly,the quantitative determination of target analytes could be achieved by the fluorescence recovery signal.On the basis of this strategy,a switch fluorescent biosensor was constructed for thr ultra-sensitive detection of nucleic acid biomarkers.1.The morphology and physicochemical properties of ZrMOF were characterized by DLS,TEM and N₂ adsorption-desorption methods.2.FTIR and Zeta potential assays were used to investigate the adsorption ability of ZrMOF toward FAM-P1/P2,and the fluorescence quenching mechanism was discussed by UV-vis absorption spectroscopy and fluorescence spectroscopy.3.The experimental conditions were optimized,for example ZrMOF amount,fluorescence quenching and recovery time.Under the optimal experimental conditions,the fluorescence spectrophotometer was utilized to quantitatively determine nucleic acid biomarkers.Results:1.ZrMOF nanoparticles were synthesized by Zr（Ⅳ）（ZrOCl₂·8H₂O）,porphyrin ligand（H₂TCPP）and benzoic acid（BA）,and the change of their particle sizes was investigated by adjusting the amount of BA.ZrMOF nanoparticles with the particle size of about 90 nm were ultimately selected,and their excellent structural properties were proved by various methods.Hereby,the switch fluorescence biosensor method was initially established based on ZrMOF as the fluorescence quencher.2.Using FAM-P1 as the specific recognition element,a ZrMOF-based switch fluorescent biosensing method was constructed for rapid and ultra-sensitive quantitative determination of DNA（T1）.Under the optimal experimental conditions,this fluorescence biosensor realized the quantitative detection of T1.In the concentration range from 1×10^-2 to 1×10~3 p M,the fluorescence intensity of FAM was linearly correlated with the logarithmic concentration of T1.The limit of detection was 2 f M（S/N=3）,and the whole detection process can be completed within 22 min.In addition,other ss DNAs with one,two,three and complete base mismatch in comparison with T1 sequence was selected as detecting interferents,which confirmed that the fluorescent biosensor showed the good specificity.3.Through changing the specific recognition element,the switch fluorescence biosensing method was also successfully applied to the rapid,ultra-sensitive and high specific detection of a nucleic acid tumor marker microRNA-21（mi R-21）.Under the optimal detection conditions,the fluorescence intensity of FAM showed a good linear relationship in the logarithmic concentration range of mi R-21(1×10^-4～10 p M).The ultra-sensitive detection of mi R-21 was achieved with a detection limit of 11 a M（S/N=3）and the whole detection time was only 27 min.The detection performance was superior to most existing biosensing methods based on other chemical materials.The good specificity of this assay was further confirmed with other four common mi RNAs.In addition,the method for mi R-21 detecting also showed the satisfactory applicability and practicability in human serum samples.The spiked recoveries were from 93.94%to 114.49%,and relative standard deviations（RSDs）were also excellently achieved from 4.23%to 6.02%.Conclusion:In this study,we prepared ZrMOF nanoparticles with excellent adsorption properties and efficient quenching characteristics.By using the specific recognition elements FAM-P1/P2,a ZrMOF-based switch fluorescent biosensor was successfully presented for rapid,ultra-sensitive and high specific detection of DNA and mi RNA.Compared with some traditional detection methods,this proposed assay possessed faster detection speed,wider detection range and lower detection limit.In addition,this method also owned the potential versatility and multiple analysis ability,and it can easily realize the detection of other target analytes only by changing to the corresponding specific recognition molecules.Hence,this strategy had potential applications and broad prospects in other fields of public health and medicine analysis.