Synthesis And Bioanalysis Research Of Lanthanide Coordination Fluorescence Probe Using Nucleotide Molecular As Ligands

The lanthanide coordination polymers?Ln-CPs?have attracted explosive attention as an emerging type of multifunctional materials in the last decades because of their unique luminescent properties,such as the high quantum yield,long luminescence lifetime,large stokes shift and narrow emission bands,as well as their intrinsic porosity,tunable composition and size.More and more Ln-CPs has been developed to detect various molecules such as the gas molecules,cations,anions,temperature/humidity and p H.However,up to now,for Ln-CPs,almost all of the organic ligands to Ln³⁺ are involved in the laborious,time-consuming synthesis progress;moreover,such organic ligands are usually poor soluble and biocompatible.Thus,the development of Ln-CPs with biological compatibility has been an important subject.As an important biomolecule,the nucleotides possess many advantages such as excellent biocompatibility,easy accessible,simple synthesis,low cost,variable structure and rich metal binding sites,especially the high affinity of phosphate groups to Ln³⁺.Based on this,in this paper,we focused on the synthesis of biocompatible Ln-CPs,using nucleotides as the organic ligand,and designed a series of biosensors for the small molecules,thrombin detection.The main work of my graduation thesis will be written in details in the sections below.1.For the first time,we synthesized a novel cerium coordination polymer nanoparticle ATP-Ce-Tris CPNs in a simple and quick way using ATP molecule as the biocompatible ligand to Ce³⁺ ions in Tris-HCl solution.In view of the excellent free radical scavenging property of cerium compounds,which is ascribed to the mixed valence state(Ce³⁺,Ce⁴⁺)and the reversible switch from Ce³⁺ to Ce⁴⁺,the synthesized ATP-Ce-Tris CPNs was used as artificial peroxidase to selectively and sensitively detect H₂O₂.Compared with those inorganic cerium oxide sensors,this kind of fluoresence ATP-Ce-Tris CPNs sensor needs no additional organic redox dye such as ABTS,TMB,or fluorescein as signal molecules,and exhibits a more sensitive response to H₂O₂ with a detection limit down to 0.6 n M.This sensing platform was further extended to the detection of glucose in combination with the specific catalytic effect of glucose oxidase?GOx?for the oxidation of glucose and formation of H₂O₂.2.It is well known,due to its strong absorption in UV region,Ce³⁺ ions have been usually used as sensitizer to transfer excitation energy to other rare earth activator in inorganic crystals.The matched energy levels of Tb³⁺?4fn?to the excited state of Ce³⁺ generated via the allowed f–d transition upon UV irradiation enables efficient energy transfer from Ce³⁺ to Tb³⁺.In order to enhance the luminescent properties of ATP-Ce-Tris CPNs,in this chapter,we synthesized a novel Ce/Tb³⁺-codoped fluorescent nanoparticle?ATP-Ce/Tb-Tris CPNs?.The synthesized ATP-Ce/Tb-Tris CPNs exhibit the characteristic green emission of Tb³⁺,for the first time we demonstrated such fluorescent lanthanide coordination polymer nanoparticles?Ln-CPNs?based on the energy transfer from metal to metal.More importantly,such nanoparticles retain numerous metal ion binding sites of ATP ligand,by which Cu²⁺ can bind to ATP-Ce/Tb-Tris CNPs readily due to its strong tendency to favor mixed N and O donor system.Considering the specific binding of Cu²⁺ ion to thiol molecule,we designed a new fluorescence probe for biothiol detection,where small amounts of Cu²⁺ were introduced to combine with ATP-Ce/Tb-Tris CPNs,then the thiol molecule bind to Cu²⁺ through its sulfur and carbonyl oxygen atoms,forming a stable ATP-Ce/Tb-Tris CPNs/Cu²⁺/Cys structure.Interestingly,the generated stable ATP-Ce/Tb-Tris CPNs/Cu²⁺/Cys structure facilitated the electron transfer from ligand to metal,giving rise to the increased fluorescence quenching of Cu²⁺ to ATP-Ce/Tb-Tris CPNs since copper ion is known as a universal fluorescence quencher.Therefore,such thiol-facilitated fluorescence quenching enables ATP-Ce/Tb-Tris CPNs/Cu²⁺ system to function as a ‘turn-off' sensor of biothiols,in which Cu²⁺ not only acts as the bridge ions,but also the electron receptor.This kind of ‘turn-off' fluorescence probe is entirely different from those analogous metal-organic complex based ‘turn-on' sensor,and exhibits much more sensitive response to biothiols.To the best of our knowledge,it is the first report on the lanthanide coordination polymer nanoparticles-based biothiols sensor,and it also provides a new strategy for biothiol sensing.As far as we known,there was no report on biomolecule detection based on the Ln-CPs.In addition,it is found that the oxidation of Ce³⁺ in ATP-Ce/Tb-Tris CNPs to Ce⁴⁺ would interrupt the energy transfer from Ce³⁺ to Tb³⁺,leading to fluorescence quenching of Tb³⁺.On the basis of this quenching mechanism,ATP-Ce/Tb-Tris CPNs has been successfully used to detect reactive oxygen H₂O₂,with detection limit as low as 2 n M.If glucose oxidase is present in the system,glucose can be determined using the ATP-Ce/Tb-Tris CNPs nanosensor.In addition,the reliability of this sensor was further evaluated by detecting the concentration of glucose in human serum samples,the good accuracy?the recovery were in the range of 97.5% to 107 %?and the high precision?RSD?demonstrated the applicability of ATP-Ce/Tb-Tris CNPs as an effective sensor for the detection of glucose in human serum.3.A lanthanide terbium coordination polymer nanoparticle?Tb-CPNs?was synthesized,using the Guanine-rich ss DNA,thrombin aptamer?TBA?as “antenna” ligands to Tb³⁺ ions in Tris-HCl buffer solution.The synthesized Tb-CPNs exhibit characteristic green emission of Tb³⁺ due to the efficient sensitization of guanine.In the presence of thrombin,the fluorescence of synthesized Tb-CPNs is quenched quickly,which may be attributed to the disintegration of the Tb-NCPs through the competitive binding of thrombin molecules with Tb³⁺.Based on this,a new sensor was developed for simple,label-free,in-situ detection of human ?-thrombin,with the detection limit down to 0.05 n M,which is comparable with those of reported aptamer-based sensors.As a practical application,the fluorescence probe was used to monitor thrombin level in human plasma with satisfactory results obtained.It provides a new strategy for the detection of thrombin.4.In order to demonstrate the mechanism of thrombin detection with Tb-NCPs as the fluorescent probe.The G-Tb CPNs?G=GMP,GDP,GTP?,which was synthesized through the self-assembly with GMP/ GDP /GTP and Tb³⁺ ions in the Tris-HCl buffer solution;the Nu-Ce/Tb CPNs?Nu =ATP,GTP,CTP,TTP,UTP?nanoparticles synthesized through the self-assembly with ATP/ GTP/ CTP/ TTP/ UTP and Ce³⁺,Tb³⁺ ions in the Tris-HCl buffer solution;the non-nucleotide ligands PO₄-Ce/Tb,P₂O₇-Ce/Tb inorganic nanoparticles,as well as the as-synthesized ATP-Ce/Tb-Tris CPNs nanoparticles were all employed to detect the thrombin.Except for the negligible fluorescence quenching of the non-nucleotide ligands PO₄-Ce/Tb,P₂O₇-Ce/Tb inorganic nanoparticles in the presence of thrombin molecules,the others Ln-CPNs with nucleotide as ligands?G-Tb CPNs,Nu-Ce/Tb CPNs,ATP-Ce-Tris CPNs?exhibit obvious response to thrombin molecules.Inaddition,the SEM,TEM and light scattering spectra of these nanoparticles upon the thrombin molecules were also carried out.The experiment results indicate that the size of all Ln-CPNs nanoparticles?G-Tb CPNs,Nu-Ce/Tb CPNs,ATP-Ce-Tris CPNs?,either with different nucleotide?A,G,C,T,U?or different emission?370nm light emitting or 550 nm light emitting?decreased gradually with increasing concentration of thrombin molecules.All results of LS,SEM,and TEM confirmed that the mechanism of thrombin detection is based on the combination between thrombin and Ln³⁺,which lead to the dissociation of this kind of lanthanide coordination polymer nanoparticles and their fluorescence quenching.It provides a new strategy for thrombin detection.