DNAzyme-mediated Fluorescence Assay Based On ZIF-8 And Its Composites For Lead Ion Detection

Lead is a heavy metal widely used in many industries such as battery manufacturing,fuel processing,and smelting.The ubiquity of lead can cause environmental pollution in air,water,soil,etc.It further accumulates in the human body through enrichment in the food chain,damaging multiple systems such as nerves,hematopoiesis,and digestion,and threatening human health.Because of this,the issue of lead contamination has received widespread attention in the fields of food safety,environmental monitoring,and biomedicine.There is an urgent need to improve existing lead detection technologies and establish new analytical methods that are convenient,sensitive,and fast.DNAzyme-based fluorescence analysis methods are widely known for their advantages such as high selectivity,high sensitivity,easy operation,and programmability.The performance can be further improved by integrating nanomaterials or nucleic acid amplification technology.Zeolitic Imidazolate Framework-8（ZIF-8）,as a kind of metal-organic framework,has the advantages of simple synthesis,low cost,high surface area,good chemical stability,and adjustable pore size.This paper studies the interface interaction between ZIF-8 and DNA,employs DNAzyme as a molecular recognition element,and uses ZIF-8 composite materials and CRISPR/Cas technology to construct three new fluorescence analysis methods for Pb²⁺detection.These methods achieve convenient,high-sensitivity,and high-specificity detection,with certain generalizability.The main research contents are as follows:（1）ZIF-8 was synthesized through static reaction at room temperature,and a variety of characterization techniques were used to prove that it has good morphology and complete structure.The differences in the adsorption of ZIF-8 to single-stranded DNA of different lengths,double-stranded DNA,and special DNAzyme structures were further studied.The results showed that the longer the length of single-stranded DNA,the higher the adsorption efficiency,while double-stranded DNA would not be effectively adsorbed.For the DNAzyme structure,if there is enough uncomplemented DNA in the loop,it can also be effectively adsorbed by ZIF-8.In addition,the mechanism of ZIF-8 adsorbing DNA is studied through potential testing and desorption experiments.The main forces of adsorption are electrostatic interactions and hydrogen bonds.The study of interface interactions lays a theoretical foundation for a deep understanding of the interaction mechanism between ZIF-8 and DNA,as well as the design of analytical methods based on ZIF-8 and DNA.（2）Based on the adsorption characteristics of ZIF-8 to DNA and the fluorescence quenching effect,a DNAzyme-mediated fluorescence assay was constructed for the detection of Pb²⁺in water and fish samples.As a fluorescence quencher,ZIF-8 has the advantages of low cost and high quenching efficiency.Pb²⁺-specific GR5 DNAzyme containing large single-stranded DNA loop can be adsorbed to ZIF-8,accompanied by fluorescence quenching of labeled FAM.After binding to Pb²⁺,GR5 DNAzyme will be cleaved from the specific cleavage site,resulting in the release of FAM-labeled single-stranded DNA containing 5 bases,thereby restoring fluorescence.This"turn-on"fluorescence method can detect Pb²⁺through a simple mixing step.It has the advantages of high sensitivity,low background,and simple operation,and is expected to be used for on-site detection of Pb²⁺.（3）The single signal output mode is more susceptible to environmental interference and instrumentation.Based on this,a ratiometric fluorescence assay based on blue fluorescent carbon dots@ZIF-8 composite material and DNAzyme was established for self-calibration of Pb²⁺detection.First,blue fluorescent carbon dots were synthesized by hydrothermal method using urea and citric acid as raw materials and were mixed and reacted with Zn²⁺and 2-methylimidazole at room temperature to prepare blue fluorescent carbon dots@ZIF-8 composite material.As a molecular recognition element,GR5 DNAzyme is adsorbed on the surface of the blue fluorescent carbon dots@ZIF-8 composite.Pb²⁺-induced DNAzyme cleavage leads to the desorption of FAM-labeled short DNA,triggering a detectable change signal in FAM fluorescence.At the same time,the self-luminescence of blue fluorescent carbon dots in the ZIF-8 composite provides a self-calibration signal.This method can achieve highly sensitive and specific detection of Pb²⁺within 60 minutes through a simple mixing process,with a detection limit of 100 p M.This method uses dual excitation wavelengths to obtain ratiometric fluorescence signals and has excellent anti-interference ability.Compared with single-function nanomaterials,the use of self-luminous multifunctional ZIF-8 composite materials can build more reliable and accurate sensors with broader application prospects.（4）With its precise recognition and trans-cleavage capabilities,the CRISPR/Cas12a system has great application potential in designing highly specific and highly sensitive fluorescent biosensors.Based on this,a ratiometric fluorescence assay using CRISPR/Cas12a and embedded red fluorescent carbon dots@ZIF-8 composite material was constructed for the detection of Pb²⁺.Red fluorescent carbon dots are encapsulated in the inner cavity of ZIF-8 to effectively shield environmental interference.Combined with the ability of ZIF-8 to adsorb DNA on its outer surface,dual fluorescence signals are established and significantly improve the accuracy of detection.The presence of Pb²⁺is converted into released activation sequences by GR5DNAzyme to activate the CRISPR/Cas12a system,which provides signal amplification through sufficient cleavage of single-stranded DNA.The method achieves high sensitivity detection of Pb²⁺with a detection limit as low as 18 p M,and has simplicity,versatility,and excellent quantitative capability.