Preparation Of Fluorescence Sensor Based On Isothermal Signal Amplification And Application In Heavy Metal Ion Detection

With the increase of high-quality living standards of human beings,heavy metal pollutant in the environment caused by human activities,containing heavy/light industry and agriculture,has been more and more serious.They are usually highly enriched and non-degradable,threatening seriously human health and the sustainable development of the biological environment.Hence,scientists have been developing a large number of excellent biosensors to detect heavy metal ions more efficiently,simply and universally.Among which,the DNA biosensors have always been the research hotspot in this field profiting from their advantages,such as low cost,strong specificity,convenient modification and convenient operation.In this paper,the T-rich oligonucleotide sequences and Cu²⁺-DNAzyme are used as the recognition elements,MCHA andλ-Exo being utilized to achieve signal amplification,which has realized respectively high sensitivity detection of Hg²⁺and Cu²⁺.The details are as follows:（1）Construction of the low background fluorescence detection system based on mismatch catalytic hairpin self-assemblyIn order to reduce the inherent background signal of traditional CHA as much as possible and improve better the signal-to-noise ratio of the above two novel fluorescent DNA biosensors,the MCHA fluorescence detection system based on molecular beacons labeled with FAM and TAMRA was firstly designed in this paper.In chapter 2,we carried out the study on the base mismatch of the partial sequence in the loop portion of the hairpin DNA（H2）,serving as the auxiliary strand of MCHA.The experimental results showed that MCHA has a higher signal-to-noise ratio than traditional CHA and achieves the highly sensitive response to the trigger chain.（2）Mismatch catalytic hairpin assembly mediated low background biosensor for efficient detection of Hg²⁺Combining the T-rich oligonucleotide sequence serving as the recognition element and the MCHA for the signal amplification,we constructed an MCHA-based fluorescent DNA biosensor for efficient detection of Hg²⁺in chapter 3.In the presence of Hg²⁺,the toe-end sequence of the hairpin DNA（H1）in MCHA were bound to the T-rich Helper DNA reserving as trigger a chain of MCHA,forming the"T-Hg²⁺-T"structure.Then,the H1 would be paired perfectly complementarily under the regulation of Gibbs free energy,launching the MCHA.The biosensor realized the efficient detection of Hg²⁺with the detection limit of 5.8n M.（3）λExonuclease-assisted cascade fluorescent DNA sensor for ultrasensitive detection of Cu²⁺Combining theλ-Exo and MCHA,the Cu²⁺-DNAzyme-based cascade DNA biosensor was developed in chapter 4.In the presence of Cu²⁺,Cu²⁺-DNAzymes were induced to undergo a hydrolysis reaction,releasing DNA fragments.Subsequently,the DNA fragment bound to the toe end of the hairpin DNA（H0）,which initiated the hydrolysis ofλ-Exo and released trigger chain of MCHA.Then the strong fluorescent signals were emitted,realizing the highly sensitive detection of Cu²⁺.Its detection limit reached 60 f M.