Novel DNAzyme-mediated Fluorescent Biosensing Methods For The Detection Of Heavy Metals Lead And Copper

Heavy metals are widely used in all walks of life,and plenty of wastes will pollute water sources,air and soil,affecting the growth of crops,plants and animals.After entering the human body through the food chain,the accumulation of heavy metals in the body will cause serious damage to human organs and nervous system.The pollution by heavy metals has become a concern in the fields of environmental monitoring,food safety and biomedicine.Thus,it is urgent to improve the existing detection technologies for heavy metals to achieve the purpose of simple operation,specific identification,rapid monitoring and accurate quantification.DNAzyme-based fluorescent biosensors own the superiority of good selectivity,high sensitivity,simplicity,rapidity,spatial visualization,multiplexing,and can flexibly integrate with nanomaterials or nucleic acid amplification technology,which helps to improve the detection performance of the method.Therefore,this thesis employed the highly specific DNAzyme as the recognition element,by means of the stable and efficient amplification technology of catalytic hairpin assembly（CHA）,introduced the nanomaterial Ti₃C₂T_X MXene with excellent performance,and based on the principle of photoinduced electron transfer（PET）and f（?）rster resonance energy transfer（FRET）.By taking Pb²⁺or Cu²⁺as model targets,four fluorescent biosensing methods were developed to achieve sensitive,simple,rapid and multiple detection.The main content and results of the research were as follows:（1）A signal-on fluorescent strategy was constructed for Pb²⁺by integrating GR-5DNAzyme,CHA and PET between G-quadruplex（G4）/hemin DNAzyme and the fluorophore.An oligonucleotide（c GRE）complementary to the enzyme strand（GRE）was used to facilitate the separation of substrate strand（GRS）from GR-5 DNAzyme.The separated GRS acted as primer to initiate the CHA process as well as the formation of G4/hemin DNAzymes,resulting in fluorescence quenching via PET.In the presence of Pb²⁺,the cleavage of GRS inhibited the CHA reaction and the generation of G4/hemin DNAzyme,accompanied by the fluorescence enhancement.This DNAzymes-CHA-PET method can detect Pb²⁺in a single tube with a linear range of 0.1-150 ng m L^-1 and a detection limit of 0.03 ng m L^-1.It exhibited the advantages of wide linear range,facile operation and good specificity.The strategy was successfully applied to detect Pb²⁺in spiked water samples and fish samples.The innovative design not only provides new ideas for the routine screening of heavy metal ions in food and environmental samples,but also opens up new avenues for the development and application of G4/hemin DNAzyme-based PET biosensors.（2）A ratiometric fluorescent biosensor was constructed for Pb²⁺analysis based on RNA cleavage-inhibited self-assembly of three-arm branched junction（TBJ）.Pb²⁺-dependent DNAzyme was employed for Pb²⁺recognition and catalytic hairpin assembly（CHA）served to form TBJ.In the absence of Pb²⁺,the primer triggered the three hairpins to form TBJ via CHA,resulting in low FAM signal and strong ROX signal.Pb²⁺-responsive cleavage of DNAzyme hindered the occurrence of CHA and the formation of TBJ,causing opposite changes in the FRET states of the two fluorophores that the FAM signal was enhanced and the ROX signal was reduced.The ratiometric fluorescence responses were recorded to indicate the concentration of Pb²⁺,endowing with more stable and reliable identification and analysis of Pb²⁺.In the concentration range of 0.05-5 ng m L^-1 for Pb²⁺,the ratio signal showed a good linear relationship with the Pb²⁺concentration,and the detection limit was 0.03 ng m L^-1.The facile method achieved the detection of Pb²⁺at constant temperature,and can afford excellent specificity for Pb²⁺against competing metal ions.The accuracy and potential applicability of the developed ratiometric biosensing method was evaluated by tea samples.This fluorescence platform provides new avenue for the development of FRET-based ratiometric approaches and DNAzyme strategies for monitoring heavy metal pollution.（3）Based on the high recognition ability of GR5 DNAzyme,the strong fluorescence quenching ability and the differences in DNA affinity of Ti₃C₂T_X MXenes,a signal-on fluorescence biosensing platform was designed to detect Pb²⁺.In the absence of Pb²⁺,Ti₃C₂T_X MXene adsorbed GR5 DNAzyme and quenched the fluorescence of FAM via FRET.In the presence of Pb²⁺,the short FAM-labeled single-stranded DNA（5-mer）was released after the cleavage of substrate strand,resulting in the fluorescence recovery of the system.Benefiting from the low background signal modulated by Ti₃C₂T_X MXenes,the sensitive determination of Pb²⁺was achieved in the linear range of 0.2-10 ng m L^-1 with a limit of detection of 0.05 ng m L^-1.The DNAzyme method exhibited the advantages of simple operation,good specificity,rapid detection（20 min）,low cost and acceptable anti-interference ability,and had been applied in the spiked water samples and SRM samples with satisfactory results.The fluorescent biosensor established in this work not only provides a theoretical basis for the DNA adsorption property of Ti₃C₂T_X MXenes,but also has great potential for on-site monitoring of lead pollution in water samples.（4）Ti₃C₂T_X MXenes presented the two-dimensional sheet-like structure,and exhibited high affinity towards FAM-labeled Pb²⁺-dependent DNAzyme（FAM-Pb-DNAzyme）and ROX-labeled Cu²⁺-dependent DNAzyme（ROX-Cu-DNAzyme）by virtue of the interaction of Ti-phosphate group.Furthermore,it can quench the fluorescence of FAM and ROX via FRET.Pb²⁺and Cu²⁺can activate the catalysis of DNAzyme and induce the cleavage of substrate chain,promoting the release of short FAM and ROX-labeled single-stranded DNA,which were not absorbed by Ti₃C₂T_X MXenes,thus showing enhanced fluorescence.Employing the two DNAzymes and Ti₃C₂T_X MXenes,the developed signal-on fluorescence method can simultaneously detect Pb²⁺and Cu²⁺by simultaneous fluorescence analysis.The detection limits of Pb²⁺and Cu²⁺was 0.03 ng m L^-1 and 0.04 ng m L^-1,respectively.The DNAzyme-based fluorescence method possessed the superiority of simple operation,low cost and high specificity.It had negligible interference from other competing metal ions or organic substances.There was no cross-reactivity between FAM and ROX probes for simultaneous detection of Pb²⁺and Cu²⁺.The simultaneous fluorescence analysis not only saves the detection time and improves detection efficiency,but also opens up new opportunity to design and develop sensing platform based on Ti₃C₂T_X MXenes and DNA.