The Detection Of Heavy Metal Lead Based On Functional Nucleic Acids Fluorescent Sensor

Lead ion(Pb²⁺)is one of the most widely polluted and most toxic heavy metal pollutants,which can accumulate in the human body through the biological chain,causing serious damage to the human body even at low concentration.Traditional detection methods have some limitations in the field of real-time monitoring due to the requirements of complex large-scale instruments and professional operators,so it is very important to develop a sensitive and simple method for rapid detection of Pb²⁺.With the rapid development of sensing technology,various fluorescent sensors based on functional nucleic acids were designed to detect heavy metal ions,toxins and proteins.In this paper,three fluorescent biosensors were constructed based on the target specific recognition and auxiliary signal amplification ability of functional nucleic acids,the rapid response of fluorescent sensor,fluorescence resonance energy transfer（FRET）effect,nuclease induced signal amplification and the applicability of paper-based sensor.The main research contents and relevant results were as follows:1.A Pb²⁺fluorescent aptasensor was constructed by using the high fluorescence quenching efficiency of gold nanoflowers（Au NFs）,the specific binding of Pb²⁺with its aptamer（Apt）and the signal amplification technology triggered by Rec Jf Exo.The aptasensor was constructed by loading SH-c DNA/FAM-Apt on Au NFs surface through Au-S bond.The high affinity binding between Pb²⁺and Apt made Apt change from random coil to G-quadruplex structure and separate from the sensor system.The disappearance of FRET between FAM and Au NFs led to the recovery of fluorescence.At the same time,the Pb²⁺was released from G-quadruplex through Rec Jf Exo reaction and circularly participated in the reaction,resulting in signal amplification.When Pb²⁺concentration was in the range of 0.5 n M-1μM,-₀has a good linear relationship with7)(₂₊),and the limit of detection（LOD）was 0.29 n M.In the experimental results,the extinction coefficient of Au NFs was 1.8 times that of Au NSs,which indicated that Au NFs has stronger fluorescence quenching ability.In addition,the sensing strategy has been successfully applied to the analysis of tap water,tea and rice powder（quality control sample）.The results showed that the developed fluorescent aptasensor provided a platform for the detection of Pb²⁺in real samples.2.The dual-signal ratiometric sensor can reduce the interference of the detection environment and avoid false positive reaction.And,the cascade signal amplification strategy can further improve the sensitivity of the sensor.Combined with Pb²⁺dependent DNAzyme and ExoⅢ-assisted cascade signal amplification,a low-noise ratiometric fluorescent biosensor was developed.The hairpin DNA（HP）labeled with Cy3 and Cy5 at both ends was used as the fluorescence probe,and the fluorescence intensity ratio of Cy3 and Cy5₅₆₂/₆₆₅was selected as response signal.The substrate chain（S-DNA）of DNAzyme was modified on the surface of magnetic beads（MBs）by biotin-SA strong binding,and then the enzyme chain（E-DNA）of DNAzyme was modified by base complementarity.In the presence of Pb²⁺,the catalytic active of DNAzyme was activated and sheared S-DNA,which released an ss DNA to open the hairpin structure of HP.At this time,the FRET effect between the two fluorophores disappeared,causing the increase of₅₆₂/₆₆₅.The ss DNA cycle was triggered by DNAzyme-ExoⅢcascade amplification strategy,which further led the signal amplification.When Pb²⁺concentration was in the range of 0.1 n M-1μM,₅₆₂/₆₆₅had a good linear relationship with7)(2+).Based on the cascade signal amplification,the sensitivity of the ratiometric biosensor was further improved,and the LOD was as low as 77 p M.The ratiometric biosensor strategy had good reproducibility and repeatability,and has also been successfully applied to the detection of water,tea and rice（quality control sample）,indicating that it had great application potential in the detection of Pb²⁺.3.The paper-based sensing platform is easy to miniaturize,simple in design and easy to carry,so it is more suitable to build high-throughput sensors for on-site detection.A high-throughput paper-based fluorescent aptamer sensor was designed for the rapid detection of Pb²⁺in whole-chain samples.The FAM-Apt-biotin chain was firstly fixed on the paper-based sensing platform through the strong combination of biotin and SA.Pb²⁺can specific bound with Apt and changed its conformation.Dabcyl-c DNA can form ds DNA with free Apt and quench the fluorescence due to the FRET effect between FAM and dabcyl.Fluorescence intensity was positively correlated with Pb²⁺concentration.When the Pb²⁺concentration was in the range of10 n M-10μM,-₀/₀had a linear relationship with7)(₂₊),and the LOD was 6.1 n M.The developed paper-based fluorescent aptasensor also realized the high-throughput detection of Pb²⁺by Microplate Reader.In addition,the method has been successfully used for the determination of Pb²⁺in lake water,soil and food samples.The paper-based sensing strategy had certain application value in the on-site rapid detection of heavy metals.