Utilizing Single OND Probe For Realization Of Detecting Trace Hg(?),Pb(?),K(?) And Cysteine,and Investigation Of The Response Mechanism

Over years,the heavy metals Hg and Pb enlightened from industry and human activities are progressively enriched in soil and water,and absorbed by the herbs and bionts through the food chain.The Hg and Pb that accumulated easily in the liver,kidney and nervous system leads to acute and chronic injuries of the digestive system,cardiovascular system,nervous system,and reproductive system.What is more,the Hg/Pb pollution was increasingly serious in the industry of Traditional Chinese Medicine(TCM)that not only lower the qualities of the crude and patent medicine but also severely threaten the effect and safety of the TCM.Thus,it becomes the objective demand for treatment of Hg/Pb pollution in the environment and TCM industry that to develop the detection method with fast,sensitivity and selectivity for the Hg/Pb contents in the samples including TCM.The established methods still exit some drawbacks in convenience,selectivity,and cost,despite their wide application.In contrast,plenty attention has focused on the approaches of fluorescence spectrum coupling specific chemical capacities of certain heavy metal ions,due to the remarkable selectivity,sensitivity,low-cost and straightforward operation.Therefore,the current work aimed to develop the method for selectively and sensitively detecting Hg(?),Pb(?),K(?)and Cysteine(Cys)with a single oligonucleotide(OND)based on the different induced secondary structures,the quenching ability of continuous two guanine bases(G₂)towards the fluorescent base HEX,the enhancing ability of parallel G-quadruplex(PGQ)towards the fluorescent ligand N-Methyl Mesoporphyrin IX(NMM),and the fluorescence recovery by Cys specific combining with Hg(?)/Pb(?).Meanwhile,the structure transfer of oligonucleotide induced by Hg(?)/Pb(?)and the quenching mechanism of G₂ to HEX in the presence of Hg(?)/Pb(?)were investigated utilizing circular dichroism spectrum(CD Spectrum),ultraviolet-visible absorption spectrum(UV-vis Spectrum),fluorescence spectrum combining with thermodynamic analysis.So as the structure transfer of oligonucleotide induced by K(?)and the enhancing mechanism of PGQ to NMM in the presence of K(?),and the mechanism of the Cys induced fluorescence recovery.The main contents are as follows:1.At first,the OND was designed as guanine bases-rich(G-rich)and thymine bases-rich(T-rich)in this work according to that the T-rich OND could fold into the Hairpin structure by the Hg(?)specifically inducing T bases to form T-Hg(?)-T mismatch and the G-rich OND could fold into the Chair-type Anti-parallel G-quadruplex(CGQ)and the PGQ induced by the Pb(?)and K(?),respectively.Then the fluorescent base HEX was labelled at the 5'-terminal of the OND.The detection of Hg(?)/Pb(?)was realized by the G₂ at3'-terminal approaching to the HEX at the 5'-terminal through the formation of the Hairpin structure or CGQ induced by Hg(?)/Pb(?)respectively,and consequently quenching the HEX through photoinduced electron transfer(PET).The detection of Cys was realized by the fluorescence recovery with the destruction of Hairpin or CGQ structure,which caused by the specific interaction of the Cys with Hg(?)/Pb(?).For K(?)detection,the fluorescence of NMM was enhanced by the PGQ,which formed from the label-free OND induced by K(?).Moreover,based on the specific interaction of NMM with PGQ and the difference between the GQ induced by K(?)and Na(I),the method of K(?)could be used for recognition and quantitative detection of K(?)in the presence of Na(I)with ultra and relatively high concentration,respectively.2.The sensitive and selective detection of Hg(?)and Cys was developed based on the formation of the Hairpin structure originated from Hg(?)specific induced T-Hg(?)-T mismatch,in the combination of the quenching ability of G₂ to HEX,as well as the interaction of Cys with Hg(?).The optimum detection parameters were as follows:PB buffer(10.0 mmol/L,p H 7.5),the concentration of probe was 20.0 nmol/L,the reaction time of Hg(?)and Cys were 10 min and 4 min,respectively.The results showed that the Hg(?)could be quantitively detected in the linear range of 10.0?140.0 nmol/L with the LOD of 8.75 nmol/L,and the linear range of 20.0?200.0 nmol/L with the LOD of 14.09nmol/L for Cys.The method performed high selectivity for Hg(?)and Cys that slight influences were observed for 15 kinds of cations in 5-fold and 12 kinds of cations in50-fold to Hg(?),as well as 10 kinds of amino acids in 10-fold to Cys.The Hg(?)detection results in three kinds of Traditional Chinese Medicine(TCM,e.g.the flower of Lonicera japonica Thunb.,the roots of Salvia miltiorrhiza Bge.and Glycyrrhiza uralensis Fisch.)by the Cold-vaper AAS method(CV-AAS)and the developed method were almost identical.And the results of the recovery experiment indicated the good application feasibility of this method in real samples.Similar conclusion for the Cys(or acetylcysteine,Acy)by comparing the detection results in fetal bovine serum and acetylcysteine granules utilizing the Tungsten blue colourimetry method and the developed method.3.The structure changes of OND induced by Hg(?)and the quenching mechanism of G₂ to HEX in the presence of Hg(?)were investigated by CD spectrum,UV-vis spectrum,fluorescence spectrum combining with thermodynamic analysis.The results showed that the introduced Hg(?)could successfully induce the designed OND forming to the Z-type Hairpin by interacting with the thymine bases into T-Hg(?)-T mismatch,and the followed introducing of Cys lead to the destruction of the Hairpin structure into random unwind coil.The quenching mechanism of G₂ to HEX was verified to be the photoinduced electron transfer(PET,one of the static quenching types)with the molar ratio of approximate 1:1,and the association was an enthalpy change dominating spontaneous process driven by Van Der Waals force and hydrogen bond.Therefore,the principle of the developed Hg(?)and Cys detection was described as follows:the Hg(?)induced the OND to form the Hairpin structure,leading to the G₂ approaching with and then quenching the HEX by PET,while the Cys induced the fluorescence recovery by interacting with Hg(?)and destructing the Hairpin structure.4.The selective detection of Pb(?)and Cys was developed based on the formation of the CGQ induced by Pb(?),in the combination of the quenching ability of G₂ to HEX,as well as the interaction of Cys with Pb(?).The optimum detection parameters were as follows:Tris-Ac buffer(50.0 mmol/L,p H 7.5),the concentration of probe was 20.0 nmol/L,the reaction time of Pb(?)and Cys were 30 min and 2 min,respectively.The results showed that the Pb(?)could be quantitively detected in the linear range of 2.0?14.0?mol/L with the LOD of 1.79?mol/L,and the linear range of 20.0-70.0?mol/L with the LOD of 6.82?mol/L for Cys.The method performed good selectivity for Pb(?)and Cys that slight influences were observed for 12 kinds of cations in 1-fold and 6 kinds of cations in 10-fold to Pb(?),as well as mere influences observed for 10 kinds of amino acids in 5-fold to Cys.The Pb(?)detection results in two kinds of TCM(the flower of Lonicera japonica Thunb.and the roots of Salvia miltiorrhiza Bge.)by the Graphite-furnace AAS method(GF-AAS)and the developed method were almost identical.The results of the recovery experiment indicated the good application feasibility of this method in real samples.Similar conclusion for the Cys/Acy by comparing the detection results in fetal bovine serum and acetylcysteine granules utilizing the Tungsten blue colourimetry method and the developed method.5.The structure changes of OND induced by Pb(?)and the quenching mechanism of G₂ to HEX in the presence of Pb(?)were also investigated by CD spectrum,UV-vis spectrum,fluorescence spectrum combining with thermodynamic analysis.The results showed that the introduced Pb(?)could successfully induce the designed OND forming to the CGQ,and the followed introducing of Cys lead to the destruction of the CGQ structure into the random unwind coil.The quenching mechanism of G₂ to HEX was verified to be the PET with the molar ratio of approximate 1:1,and the association was an enthalpy change-dominating spontaneous process driven by Van Der Waals force and hydrogen bond.The verified quenching mechanism in the presence of Pb(?)was identical to the Hg(?).Therefore,the principle of the developed Pb(?)and Cys detection was described as follows:the Pb(?)induced the OND forming to the CGQ structure,leading to the G₂approaching with and quenching the HEX by PET,while the Cys induced the fluorescence recovery by interacting with Pb(?)and destructing the CGQ structure.6.The highly sensitive and selective detection of K(?)was developed based on the formation of the PGQ induced by K(?)in combination with the enhancement ability of PGQ to the fluorescence of NMM.The optimum detection parameters were as follows:Tris-Ac buffer(50.0 mmol/L,p H 7.5),the concentration of probe was 2.0?mol/L,the concentration of NMM was 1.0?mol/L,the reaction time was 40 min.The results showed that the K(?)could be quantitively detected in the linear range of 5.0?30.0?mol/L with the LOD of 1.28?mol/L.The method performed high selectivity that mere influences were observed for 9 kinds of cations in 1-fold to K(?),despite the slight influence of Ca(?).Moreover,it was verified that the developed method could be utilized in recognition and quantitively detection for K(?)in the presence of Na(I)with concentrations of at least5000-fold and 600-fold,respectively.7.The structure changes of OND induced by K(?)and the enhancement mechanism of PGQ to the fluorescence of NMM were investigated by CD spectrum,fluorescence spectrum in combination with thermodynamic analysis.The results showed that the introduced K(?)could successfully induce the designed OND forming to the PGQ structure.In contrast,the Na(I)could only weakly induce OND forming to an un-chair type anti-parallel G-quadruplex that NMM did not favor.The association of the PGQ to NMM was verified to be an entropy change-dominating spontaneous process driven by hydrophobic force,with the molar ratio of approximate 1?2:1.The enhancement mechanism of PGQ to NMM was speculated as that the inner hydrophobic microenvironment of PGQ protected the fluorescence of NMM from the strong influence of solution ion.Therefore,the principle of the developed K(?)was described as follows:the PGQ structure,which formed form OND by the K(?)inducing,combined with NMM,and protected it from ion's influence by hydrophobic force,leading to the fluorescence enhancement.And the K(?)recognition and detection under high Na(I)concentration was realized based on the differences of inducing abilities and the induced GQ formations between Na(I)with K(?).