Studies On New Method For Detection Of Mercury Ions And Lead Ions With Functional Nucleic Acid Probes

Purpose:With the increase of human industrial activities,the problem of heavy metal pollution in the environment has been widely concerned and highly valued.Heavy metal pollution is persistent,accumulative and irreversible,which can have long-term and far-reaching effects on the environment and seriously threaten human health.The monitoring of heavy metal ions such as lead(Pb²⁺)and mercury(Hg²⁺),which are commonly found in the environment,has always been the focus of the comprehensive management of heavy metal pollution.Based on traditional detection methods of heavy metal ions,the continuous development of new methods for rapid and accurate detection using new materials,which is an opportunity and challenge for the development of scientific and technological innovation in the field of environmental health.And it is of great significance for the prevention and control of heavy metal pollution.Nucleic acid probes have the advantages of real-time,intuitive,convenient and sensitive.They are widely used in analysis and detection in many fields such as biomedicine,environment and food,and have broad development prospects.Using functional nucleic acids with high specificity as sensing elements to construct and design probes for heavy metal ion detection,which can show good performance.Therefore,this study is based on two functional nucleic acids,metal ion-specific base pair(T-Hg²⁺-T)and DNAzyme,combined with novel micro-nanomaterials to design and construct nucleic acid probe sensors for Hg²⁺and Pb²⁺,respectively.And explore the detection application performance of the probe in the environment or cells.It provides a new strategy for realizing rapid,sensitive and visual identification and detection of heavy metal ions,and to provide new ideas for the research and development of heavy metal ion monitoring technology.Methods:1.A novel dual-mode detection method for Hg²⁺based on T-Hg²⁺-T specific base pair combined with Au NRs and MPFs@Ru（dcbpy）₃²⁺（1）Preparation and characterization of Au NRs and MPFs@Ru（dcbpy）₃²⁺Firstly,Au NRs with uniform size were prepared and purified by a typical seed-mediated growth process using chloroauric acid（HAu Cl₄）as raw material.The particle size and morphology of Au NRs were characterized by scanning electron microscopy,transmission electron microscopy,and ultraviolet visible spectroscopy.In addition,the metal-organic framework ZIF-8 was synthesized by a simple one-step method using zinc nitrate（Zn（NO₃）₂·6H₂O）and dimethylimidazole（2-MI）as raw materials.Then,ZIF-8 was used as template to synthesize hollow metal-polydopamine frameworks（MPFs）with abundantπelectrons.Ruthenium terpyridine can be modified on MPFs byπ-πstacking reaction to form MPFs@Ru（dcbpy）₃²⁺complexes.Fourier transform-infrared spectroscopy,scanning electron microscopy,transmission electron microscopy,X-ray photoelectron spectroscopy,ultraviolet-visible spectroscopy,etc.were used to characterize the changes of morphological structure,chemical element and characteristic absorption peaks of MPFs@Ru（dcbpy）₃²⁺composites.（2）Preparation of DNA1-coated magnetic beads（MBs-DNA1）and alkaline phosphatase-conjugated DNA2（ALP-DNA2）probesBiotin-labeled T-rich DNA1 and DNA2 were designed.Through the specific binding between avidin and biotin,the MBs-DNA1 and ALP-DNA2 probes were prepared by linking with avidin-modified MB and ALP respectively,which were used as sensing elements for recognizing Hg²⁺.（3）Feasibility verification of dual-mode sensing analysis and detectionALP can hydrolyze ascorbic acid phosphate（AAP）to produce ascorbic acid（AA）.In the electrochemiluminescence（ECL）sensing analysis platform,ECL technology was used to study the luminescence mechanism of MPFs@Ru/S₂O₈^2-system and the AA quenching luminescence signal mechanism.In the colorimetric sensing analysis platform,the enzymatic hydrolysis product AA can be used as a reducing agent to reduce silver ion to silver deposited on Au NRs to form Au NRs@Ag.To demonstrate the feasibility of Ag deposition on Au NRs,the control experiment was designed to observe the color change of the reaction solution with the naked eye.And local surface plasmon resonance（LSPR）peak changes can be measured for analysis.TEM images were used to verify the morphology change of Au NRs after Ag deposition.（4）Research on response characteristics of dual-mode sensing analysis PlatformFirst,the prepared MBs-DNA1,ALP-DNA2 and Hg²⁺were hybridized using the T-Hg²⁺-T mismatch mechanism.After magnetic separation and washing,the MBs-DNA1/ALP-DNA2 complexes were added to the wells of 96-well plates.AAP was added to carry out the enzymatic catalyzed dephosphorization reaction to AA.Then,a part of the reaction solution was transferred to the electrolytic cell to detect the ECL signal.Another part of the solution was transferred to the mixture containing Ag NO₃ and Au NRs,and the color change by the deposition of Ag on Au NRs was reduced by AA for colorimetric detection.Explore the response properties of different mechanisms and independent signal transduction modes.（5）Analytical application in real environment samplesLake water samples were collected,and the new dual-mode sensing method designed in this study was used to detect the water samples to evaluate the feasibility of this method for mercury ion detection in the actual environment.2.A new method for Pb²⁺fluorescence imaging detection based on DNAzyme combined with worm-like core-shell nanochains（Au@PDA NWs）（1）Synthesis and characterization of Au@PDA NWsFirstly,gold nanoparticles（Cit-Au NPs）with a size of about 40 nm were synthesized by using citrate as a reducing agent and the method of kinetically controlling the growth of seeds.The morphology and properties of Cit-Au NPs were demonstrated and analyzed by transmission microscopy and UV-vis spectrum methods.Then,using dopamine hydrochloride solution as raw material,under the condition of alkaline p H,polydopamine（PDA）shells are aggregated on the surface of gold nanoparticles through ultrasonic reaction to form core-shell nanoparticles.The morphology,crystal structure,and properties of Au@PDA NWs were characterized by transmission electron microscopy,field emission transmission electron microscopy,X-ray photoelectron spectroscopy,X-ray diffraction,and UV-vis spectroscopy.（2）Construction and characterization of DNAzyme nanoprobesThe designed Pb²⁺-specific DNAzyme was heated and cooled to form hairpin-like DNAzyme（H DNAzyme）.The hairpin DNAzyme probe was successfully loaded into Au@PDA NWs through coordination andπ-πstacking to form Au@PDA NWs-H DNAzyme nanoprobes.The construction of the probe was verified by zeta potential analysis and fluorescence spectroscopy.（3）Response performance verification of constructed nanoprobesThe response properties of nucleic acid probes to lead ions was verified using native polyacrylamide gel electrophoresis,and imaged with gel imaging system for analysis.The feasibility of the constructed probe to detect lead ions was verified by fluorescence spectroscopy.The constructed nanoprobes were mixed with lead ion solutions of different concentrations.After reacting for a period of time,and the supernatant was collected by centrifugation for fluorescence test.The change of fluorescence intensity was observed to test the response performance of the designed probe to Pb²⁺in the solution.（4）Fluorescence imaging analysis of Pb²⁺in cells and zebrafishHela cells were utilized throughout the experiment.And the performance of nanoprobes in recognizing Pb²⁺in living cells was further tested.The MTT Kit was used to evaluate the cytotoxicity of Au NP@PDA NWs on Hela cells.After the cells were incubated with Pb²⁺,the probes were delivered into the cells for adequate uptake.Finally,the cells were visualized by confocal microscopy.The performance of designed nanoprobes for imaging metal ion in living cells was verified by control experiments to observe the fluorescence intensity changes.In addition,in order to further demonstrate the ability of the nanoprobe to image lead ions in animals,the zebrafish larvae exposed to Pb²⁺was incubated with nanoprobes.Confocal microscopy was used to observe and record the imaging of Pb²⁺in zebrafish larvae.Results:1.A novel dual-mode detection method for Hg²⁺based on T-Hg²⁺-T specific base pair combined with Au NRs and MPFs@Ru（dcbpy）₃²⁺（1）Preparation and characterization of Au NRs and MPFs@Ru（dcbpy）₃²⁺Transmission electron microscopy showed that the Au NRs had a dispersed and uniform nanorod-like structure,and the color of the material solution was pink.The UV-vis spectra showed that Au NRs had a longitudinal plasmon resonance absorption peak at 860 nm.The synthesized ZIF-8 was observed by field emission electron microscopy as a nanocube structure of approximately 500 nm.After the reaction with dopamine hydrochloride,the synthesized MPFs showed hollow structure with rough surface.The HAADF-STEM image also verified the result.At the same time,the absorption peaks and elemental compositions of the synthesized materials were analyzed and compared by Fourier transform infrared spectroscopy,UV-vis spectroscopy and X-ray energy spectroscopy,indicating that the successful synthesis of MPFs@Ru（dcbpy）₃²⁺.（2）Mechanism verification of dual-mode sensing analysis detection methodBy forming T-Hg²⁺-T base pairs,MBs-DNA1 and ALP-DNA2 can specifically capture Hg²⁺pairing as duplex probes.ALP collected by magnetic separation,which induces AAP to produce AA.The obvious decrease of the response ECL signal indicates that AA can quench the luminescence signal of MPFs@Ru/S₂O₈^2-system.We speculated that HA^-would compete with Ru（dcbpy）₃⁺or Ru（dcbpy）₃²⁺to react with the crucial substance SO4^·-in the ECL system,thereby reducing the fnal excited state substances and achieving a quenching effect.In the colorimetric sensing analysis system,it was observed that the surface of Au NRs was wrapped with a thin shell by transmission electron microscopy,accompanied by the color change from pink solution to green solution.UV-Vis spectroscopy found that the LSPR peak was blue-shifted.These results demonstrate that AA can reduce Ag NO₃ to deposit silver shells on Au NRs.（3）Response performance verification of dual-mode sensing analysisImportant conditions affecting the experiment were optimized,including the concentration of K₂S₂O₈,the p H value of the detection solution,the incubation time of the T-Hg²⁺-T reaction and the hydrolysis time of ALP.It was found that the optimum concentration of K₂S₂O₈ was100 mmol L^-1,the optimum p H was 8,the optimum reaction time of T-Hg²⁺-T was 60 min,and the optimum hydrolysis time of ALP was 20 min.Under this optimal experimental condition,different concentrations of mercury ions were detected and analyzed.The color of the solution changed vividly from pink to orange,yellow,cyan,green,and black green with the increase of Hg²⁺.The dual-sensing assay determined that the signal response value had a good linear relationship with the concentration of Hg²⁺in the range of 2 pmol L^-1 to 500 nmol L^-1,and the detection limit was as low as 0.32 pmol L^-1.The method exhibits good selectivity,stability and reproducibility.The colors were transformed into RGB values,which were obtained by Color Picker installed in the smartphone.Meanwhile,the color change can also be transduced into the change in the R/G value.The R/G value decreased with increasing Hg²⁺concentration,indicating that the R/G value can be utilized as a parameter for Hg²⁺quantifcation.And it showed a good linear correlation,indicating that this method can be used to detect Hg²⁺in a portable way using smartphone.（4）Research on detection in actual environment samplesBased on ECL sensing analysis,the recovery of spiked samples in lake water ranged from 98.53%to 111.97%,and the colorimetric method was 95.04%to 106.11%,indicating that this method has the potential to on-site instant detection of Hg²⁺in actual environmental water samples.2.A new method for Pb²⁺fluorescence imaging detection based on DNAzyme combined with worm-like core-shell nanochains（Au@PDA NWs）（1）Preparation and characterization of Au@PDA NWsUV-vis spectra analysis found that Au@PDA NWs exhibit an absorption peak around 800 nm,indicating that Au@PDA NWs could effectively absorb near-infrared light.The nanoworm-like structure of Au@PDA NWs was clearly seen by transmission electron microscopy It can be observed that the Au@PDA NWs consist of 4 to 5 gold nanoparticle cores wrapped by PDA shell,and the average dimension of Au@PDA NWs was about 200 nm length.The element mapping map effectively verified the existence of the core-shell structure of Au@PDA NWs.The elemental composition of Au@PDA NWs was verified by Fourier transform infrared spectroscopy,X-ray photoelectron spectroscopy,and X-ray diffraction patterns,and the results showed that Au@PDA NWs were successfully synthesized.（2）Validation of the response mechanism of the Au@PDA NWs-H DNAzyme probeThe cleavage efficiency of the designed H DNAzyme to Pb²⁺was observed by polyacrylamide gel electrophoresis imaging.With the increase of reaction time,the cleavage reaction efficiency gradually increased and reached plateau at 30 min.At the same time,the cleaving efficiency of different concentrations of lead ions has also been proved.With the increase of lead ion concentration,the cleavage reaction is gradually enhanced,confirming that the cleavage reaction is concentration-dependent.All the above results indicate that H DNAzyme has excellent catalytic activity towards Pb²⁺and can effectively mediate the cleavage reaction.Then,it was confirmed that the hairpin DNAzyme could be effectively loaded into Au@PDA NWs by the negative change of Zeta potential,indicating that the Au@PDA NWs-H DNAzyme probe was successfully constructed.Fluorescence spectroscopy was used to verify the response of Au@PDA NWs-H DNAzyme probe to Pb²⁺,and it was found that the fluorescence signal of the collected supernatant was enhanced,indicating that the construction of the probe can realize the response strategy of"turn-on"fluorescence signal in the presence of Pb²⁺.（3）Design probe for in vitro sensing response performance verificationThree important experimental conditions were optimized,including the immobilization time of H DNAzyme to Au@PDA NWs,the concentration of the Ca²⁺buffer,and the incubation time with Pb²⁺solution.60 min was selected as the optimal incubation time,and the optimal concentration of Ca²⁺in buffer solution was 6 m M and the optimal incubation time with Pb²⁺solution was 30 min.Under the optimal experimental conditions,lead ions with different concentrations were detected and analyzed.The fluorescence response signal is linearly correlated with the concentration of Pb²⁺in the range of 0.01-20 n M.These result suggested that the Au@PDA NWs-H DNAzyme nanoprobe can be effectively used for the sensitive in vitro detection of Pb²⁺.（4）Fluorescence imaging detection of lead ions in cells and zebrafish larvaeThe results of cytotoxicity experiments showed that Au@PDA NWs had good biocompatibility and low toxicity.We optimized the cellular uptake time of the nanoprobes and selected 3 h as the optimal cellular uptake time.Confocal fluorescence images showed that Au@PDA NWs-H DNAzyme could effectively enter the intracellular environment,and no obvious background fluorescence was observed.After Hela cells incubuted with different concentrations（100 n M,500 n M,5μM）of Pb²⁺,the nanoprobes were used for imaging detection and analysis.It was found that the red fluorescence intensity increased with the increase of Pb²⁺concentration.The above results indicate that the Au@PDA NWs-H DNAzyme nanoprobe can be used for imaging and analysis of Pb²⁺in living cells.In addition,nanoprobes was performed on Pb²⁺-treated 3 days post fertilization（dpf）larvae for imaging Pb²⁺in vivo.Compared with the control group,the fluorescence intensity of the Pb²⁺-treated group was enhanced and the accumulation of red fluorescence was observed,suggesting that the probe also has the potential to imaging Pb²⁺in vivo.Conclusion:In this study,using functional nucleic acid probes and different novel materials,based on three sensing and analysis techniques（colorimetric method,ECL method and fluorescence method）,the dual-mode detection of Hg²⁺and the imaging detection of Pb²⁺were realized respectively.The designed new dual-mode detection method for Hg²⁺effectively combines the two sensing modes,which can detect Hg²⁺quickly and accurately.Combined with the smart phone and multi-color analysis method,a more convenient on-site real-time visual detection of Hg²⁺is realized through the smart phone APP,which achieves both qualitative and quantitative detection of heavy metal Hg²⁺polluted ions in the water environment.The designed new method for Pb²⁺fluorescence imaging detection can clearly image Pb²⁺in living cells or zebra larvae,which has the potential value of further realizing the application of in vivo sensing and detection.In conclusion,this study provides a feasible strategy for promoting the on-site instant detection of heavy metal pollution in the development environment and the technological innovation and development of visual imaging detection of heavy metal ions in living cells.And it provides an experimental basis for better prevention and control of heavy metal pollution.