Detection Of Heavy Metal Ions And Thrombin Based On Silver Nanocluster Fluorescent Probes

Fluorescence analysis has been applied in different research fields and played the extremely important roles in medical diagnosis,pathological research,and environmental monitoring,etc.With the flourish development of nanoscience and nanotechnology,various fluorescent nanomaterials with different morphologies have been successfully synthesized.In recent years,nobel metal nanoclusters have attracted more and more attentions as a new type of fluorescent nanomaterials.The silver nanoclusters(AgNCs)have especially attracted great interests because of their unique properties such as simple synthesis methods,ultra-small size,good biocompatibility and low cytotoxicity.With the rapid development of modern society and economy,environmental pollution becomes increasingly serious.Heavy metal ions can particularly causeserious damage at low concentrations.Therefore,it is very important to establish a rapid and sensitive method to detect heavy metal ions.Thrombin can catalyze many coagulation-related biological reactions,such as thrombosis and hemostasis.Excessive thrombin will induce thrombosis while low content of thrombin will induce an excessive bleeding.Therefore,it is also important to establish a rapid and sensitive method for the detection of thrombin.In this thesis,new methods have been developed for detection of heavy metal ions and thrombin by using AgNCs as fluorescent probe,respectively.It consists of four chapters:In the introduction chapter,the progress of different synthesis methods and applications of DNA-AgNCs were firstly introduced.Then,the methods of detecting heavy metal ions with fluorescent probes in recent years were reviewed,and the advantages and disadvantages of various probes were also sumarized.Finally,the importance of proteases and different detection methods were introduced.In the second chapter,stable silver nanoclusters with high quantum yields were synthesized by using the terminal thiol-containing DNA chains.Both Hg2+ and Cu2+can quench the fluorescence of the silver nanoclusters,then be used to detect these two metal ions.The silver nanocluster solution were added with different concentrations of mercury ions or copper ions,which resulted in that the fluorescence gradually decreased.By using the new dC12-SH-AgNCs fluorescent probe,it was analyzed wtih Hg2+ across a dynamic range of 0.5?3000 nM and a minimum detectable concentration of 0.3 nM.The detection range of Cu2+ is 1.0?3000 nM and the minimum detectable concentration is 0.7 nM.In addition,it is also possible to selectively detect mercury ions by adding a chelating agent EDTA to mask copper ions,indicating that this method can be applied to detect mercury ions in complex systems.In the third chapter,DNA-AgNCs and N-methyl mesoporphyrin IX(NMM)were used as dual-signal fluorescent probes to establish a method for detecting thrombin through double-enhanced fluorescence signals.The methods can reduce false positive results and increase the reliability of results.Three DNA strands are designed in the detection method:one of which contains a DNA strand(cDNA)complementary to the thrombin aptamer,the second one is 15-mer thrombin aptamer strand(TBA),and the third one is G-sequence-rich DNA strand(Ag-En).The principle of detection of thrombin is:DNA-AgNCs was firstly synthesized by using cDNA as template through the reduction AgNO3 by NaBH4.Then,thrombin aptamers(TBA)and NMM was added to form dsDNA-AgNCs-NMM.In this case,the fluorescence signals of AgNCs and NMM were very weak.As thrombin was added to the above solution and incubated,thrombin and its aptamer specifically combined together to form a G-quadruplex.Thus,NMM was embedded in the G-quadruplex and fluorescence was greatly enhanced.At the same time,G-sequence-rich DNA(Ag-En)and cDNA were complementarily paired to form dsDNA-AgNCs,which resulted in the approaching of G-sequence-rich DNA to the synthetic DNA-AgNCs,and enhancement of the fluorescence of AgNCs.The quantitative relationship between the double-enhanced fluorescence signal and thrombin concentration was established.The detection range of fluorochrome NMM for thrombin was 0?1.0 ?M and the detection limit was 4.5 nM and detection range of silver nanocluster for thrombin was 0?1.0?M,and the detection limit was 9.3 nM.The advantage of this method is high reliability of the detection results,label-free,reduced cost and the simple operation.In the fourth chapter,the fluorescence resonance energy transfer system was constructed by using AgNCs and fluorescein isothiocyanate(FITC)-labeled peptides to detect thrombin activity.A peptide substrate with three-part sequence was designed,which consists of the labeled FITC at N-terminus,thrombin recognition sequence and cysteine residue as C-terminus.The designed polypeptide is attached to AgNCs by the sulfhydryl group in the cysteine residue so that the FITC donor undergoes fluorescence resonance energy transfer to AgNCs receptor.The fluorescence of AgNCs was enhanced,and the fluorescence of FITC was weakened.When thrombin is added,thrombin hydrolyzes the peptide chain into amino acid fragments.Therefore,the distance between FITC and AgNCs becomes lager and the fluorescence resonance energy transfer system is destroyed,which resulted in that the fluorescence of AgNCs decreases and the fluorescence of FITC increases.Thus,the activity of thrombin is detected according to the change of fluorescence signal.The linear range of detection for thrombin activity was 8.5?34 mU/L,and the detection limit was 5.1 mU/L.This work will provide the feasibility to the energy transfer properties of AgNCs in biological/chemical sensing.