The Study On Fabrication Of Fluorescent Probe Based On Conjugated Fluorene And Functional Nucleic Acids And Its Application In Detecting Lead Ion

As a heavy metal ion that widely existed in environment,lead ion is environmental pollution because of its high toxic,bioaccumulative and nonbiodegradable.The lead can be absorbed by human body through diet,and easily to cause lead poisoning and threaten the life after enriching to a certain level.The traditional instrumental methods require expensive equipment and complicated sample preparation process.Therefore,it is of great significance to construct a novel analysis method for Pb²⁺detection of simple operation,high sensitivity and low cost.Biosensors with functional nucleic acids as recognition units have been attracted a great attention in the analytical field due to their high sensitivity and specificity.Fluorescence analysis are widely used in environmental monitoring,biological imaging,molecular diagnostics and other fields due to its high sensitivity and good reproducibility.Fluorescent nucleic acid probe is constructed by functional nucleic acids having high affinity and high specificity binding with the target as recognition unit,and a fluorescent material is capable of emitting fluorescence as a signal unit.The conjugated fluorene has stable luminescence performance and high quantum yield.In this thesis,a single-labeled functional nucleic acid probe?CF-DNA?was designed with a conjugated fluorene derivative?CF?as signal unit,which simplified the synthesis steps and avoided the negative influence of complex markers on specific recognition ability.The obtained fluorescent probe presented high sensitivity and selectivity for detecting Pb²⁺which showed remarkable innovation,and the results are listed as follows:?1?2,7-bis?4-phenylcarboxylic acid methyl ester?-9,9-di-n-octylfluorene?CF1?was synthesized by 2,7-Dibromo-9,9-di-n-octylfluorene as a raw material,and then was hydrolysis to obtain 2,7-bis?4-phenylcarboxylic acid?-9,9-di-n-octylfluorene?CF₂?.CF₂ was then reacted with N-hydroxysuccinimide to prepare conjugated fluorene active ester?CF?.All the structures were confirmed by FT-IR and ¹HNMR spectra.CF-DNA probe was obtained by reaction of CF with amino modified G-rich sequence Pb²⁺-DNA,and purified through HPLC.The CF-DNA probe had distinct UV absorption peaks at 260 and 340 nm,and the molecular weight is 5497.4,which was consistent with the theoretical design value.The fluorescence excitation wavelength of CF-DNA probe was 340 nm,and the fluorescence emission wavelength was 415 nm.?2?With the addition of Pb²⁺,the positive peak at 260 nm and the negative peak signal at240 nm were significantly enhanced in circular dichroism analysis.The particle size of CF-DNA colloidal particles in aqueous solution reduced from 1254 nm to 1003 nm.It indicated that nucleic acid molecule changed from a disordered state to a parallel G-quadruplex structure,and the CF-DNA probe had good specific binding to Pb²⁺.The addition of Pb²⁺changed the aggregation state of CF-DNA probe in aqueous solution.The optimized condition for Pb²⁺detection was:the concentration of CF-DNA probe was 100 nM,PBS buffer solution was selected as the buffer solution,the reaction time of CF-DNA probe with Pb²⁺was 15 min,and the reaction temperature was 30°C.?3?The CF-DNA probe exhibited high sensitivity to Pb²⁺detection with a detection limit of 0.36 nM,and provided excellent selectivity for Pb²⁺detection.The CF-DNA probe showed good performance in repeatability and stability.In the Pb²⁺detection of actual water samples by spiked recovery method,the recovery was in a range of 96.6%-106%,presenting the great detection effect.The CF-DNA probe had great application prospect for Pb²⁺detecting in environmental samples.?4?The CF-DNA/GO probe was constructed with the CF-DNA probe and graphene oxide?GO?,based on the properties of GO that can strongly absorb single-stranded DNA and has high efficiency fluorescence quenching performance.The optimal conditions for detecting Pb²⁺by CF-DNA/GO probe were:CF-DNA probe concentration was 150 nM,GO concentration was50?g/mL,the reaction time of CF-DNA/GO probe with Pb²⁺was 30 min,and the reaction temperature was 30°C.?5?The CF-DNA/GO probe exhibited high sensitivity to Pb²⁺detection with a detection limit of 0.24 nM,and provided excellent selectivity for Pb²⁺detection.The CF-DNA/GO probe showed good performance in repeatability and stability.In the Pb²⁺detection of actual water samples by spiked recovery method,the recovery was in range of 98.3%-102%,presenting the great detection effect.The CF-DNA/GO probe had great application prospect for Pb²⁺detecting in environmental samples.