| The fluorescence assay is used in environmental monitoring and life science more and more widely due to their excellent sensitivity and good selectivity. In the early days of the fluorescence assay, the organic fluorescent dyes used have the disadvantages of narrow excitation spectra, broad and asymmetry emission spectra, and poor photostability. In addition, the fluorescence spectra of the organic fluorescent dyes probes are usually overlapped with the intrinsic fluorescence of biological systems, resulting high background signal, low sensitivity, and vulnerable to environmental impacts. These weaknesses affect the application of the organic fluorescent dyes based fluorescence assays. Hence, it is important to synthesis novel long life-time fluorescent materials, and eliminate the background signal of samples and solid substrate using time-gated methods. This paper mainly synthesis a series of long life-time dyes with excellent luminescent properties, and then use the synthesized dyes for the detection of heavy metal ions in the environment, biomolecules, and trace water in the organic solvents. The detection methods proposed in the paper have the advantages of high sensitivity and good selectivity.This paper has completed the following work:(1) The authors herein described a time-gated fluorescence resonance energy transfer sensing strategy employing water-soluble long lifetime fluorescence quantum dots and gold nanoparticles to detect trace Hg2+ions in aqueous solution. The water-soluble long lifetime fluorescence quantum dots and gold nanoparticles were functionalized by two complementary ssDNA except for four deliberately designed T-T mismatches.The quantum dot was acted as the energy transfer donor, and the gold nanoparticle was acted as the energy transfer acceptor. When Hg2+ions were present in the aqueous solution, DNA hybridization will occur due to the formation of T-Hg2+-T complexes. As a result, the quantum dots and gold nanoparticles are brought into close proximity, which made the energy transfer occurred from quantum dots to gold nanoparticles, leading to the fluorescence intensity of quantum dots decreased obviously. The decrement fluorescence intensity is proportional to the concentration of Hg2+ions. Under the optimum conditions, the sensing system exhibits a same liner range from1×10-9mol/L to1×10-8mol/L for Hg2+ions with the detection limits of0.49nmol/L in buffer and0.87nmol/L in tap water samples. This sensor was also used to detect Hg2+ions from samples of tap water, river water and lake water spiked with Hg2+ions, and the results shown good agreement with the found values determined by Atomic Fluorescence Spectrometer. The sensor also exhibits excellent selectivity (chapter2).(2) An ultrasensitive "turn-on" fluorescent sensor was presented for determination of Hg2+. This method is mainly based on Hg2+-induced conformational change of a thymine-rich single-stranded DNA. The water-soluble long lifetime fluorescence quantum dot (Mn:CdS/ZnS) acted as the fluorophore, which was labeled on a33-mer thymine-rich single-stranded DNA (probe2). The gold nanoparticles (GNPs) functionalized10-mer single-stranded DNA (probe1) is selected as the quencher to quench the fluorescence of Mn:CdS/ZnS. Without Hg2+in the sample solution, probe1and2could form hybrid-structures, resulting in the fluorescence of Mn:CdS/ZnS decreased sharply. When Hg2+is present in the sample solution, Hg2+-mediated base pairs induced the folding of probe1into a hairpin structure, leading to the release of GNPs-tagged probe2from the hybrid-structures. The fluorescence signal is then increased obviously compared with that without Hg2+Meanwhile, a detection limit of0.18nmol/L is estimated based on3a/slope. Selectivity experiments reveal that the fluorescent sensor is specific for Hg2+even with interference by high concentrations of other metal ions. This sensor is successfully applied to determination of Hg2+in tap water and lake water samples (chapter3).(3) a sensitive time-gated fluorescent method for mercury ions (Hg2+) monitoring is developed based on Hg2+-mediated thymine (T)-Hg2+-T structure and the mechanism of fluorescence resonance energy transfer from Mn-doped CdS/ZnS quantum dots to graphene oxide. The authors employ two T-rich single-stranded DNA (ssDNA) as the capture probes for Hg2+, and one of them is modified with Mn-doped CdS/ZnS quantum dots. The addition of Hg2+makes the two T-rich ssDNA hybrids with each other to form stable T-Hg2+-T coordination chemistry, which makes Mn-doped CdS/ZnS quantum dots far away from the surface of graphene oxide. As a result, the fluorescence signal is increased obviously compared with that without Hg2+The time-gated fluorescence intensities are linear with the concentrations of Hg2+in the range from0.2to10nmol/L with a limit of detection of0.11nmol/L. The detection limit is much lower than the U.S. Environmental Protection Agency limit of the concentration of Hg+for drinking water. The time-gated fluorescent method is specific for Hg2+even with interference by other metal ions. Importantly, the proposed method is applied successfully to the determination of Hg2+in natural water samples (chapter4).(4) Gold nanoparticles (GNPs) can effectively differentiate the unfolded and folded aptamer, and quench the fluorescence of terbium ternary complexes (Tb-complexes), thus the authors herein report a sensitive strategy for protein detection, using label-free aptamer, Tb-complexes and GNPs. In the presence of thrombin, the aptamer is inclined to form G-quartet, and the folded aptamer cannot adsorb on the surface of GNPs, induced the GNPs aggregation in the presence of0.5mol/L salt. After centrifugation at low speed to remove the aggregated GNPs, the quenching capability of the supernatant for Tb-complexes is decreased. The fluorescence intensity of Tb-complexes is increased with the concentration of thrombin increased. Due to the highly specific recognition ability of the aptamer for thrombin and the strong quenching property of GNPs for Tb-complexes, the proposed protocol has good selectivity and high sensitivity for thrombin. Under the optimum conditions, a linear range from1.0×10-9mol/L to1.0×10-8mol/L is obtained with a detection limit of0.14nmol/L, which is much lower than those commonly used colorimetric sensors and some fluorescent sensors. The proposed sensor has been successfully applied in complicated biological samples for thrombin detection (chapter5).(5) In the present study, the authors report a novel sensitive method for the detection of thrombin using time-resolved fluorescence sensing platform based on two different thrombin aptamers. The thrombin15-mer aptamer as a capture probe was covalently attached to the surface of glass slide, and the thrombin29-mer aptamer was fluorescently labeled as a detection probe. A bifunctional europium complex was used as the fluorescent label. The introduction of thrombin triggers the two different thrombin aptamers and thrombin to form a sandwich structure. The fluorescence intensity is proportional to the thrombin concentration. The present sensing system could provide both a wide linear dynamic range and a low detection limit. The proposed sensing system also presented satisfactory specificity and selectivity (chapter6).(6) In this protocol, the authors report a time-resolved fluorescence biosensor based on home-made europium complexes for highly sensitive detection of small molecules using adenosine as a model analyte. The fluorophore that used is europium complexes. Its signal can be measured in a time-resolved manner that eliminates most of the unspecific fluorescent background. The amino modified aptamer probe, which is designed to specifically recognize adenosine, is combined to the aldehyde-group modified glass slide by covalent bond. Europium complex-labeled a short ssDNA, designed to segment hybridize with aptamer probe is immobilized on the glass slide by hybridization reaction. In the presence of adenosine, the aptamer part is more inclined to bounds with adenosine and triggers structure-switching of the aptamer from aptamer/ssDNA duplex to aptamer/target complex. As a result, europium complexes-labeled ssDNA is forced to dissociate from the sensor interface, resulting in time-resolved fluorescence intensity decrease. The decrement intensity is proportional to the amount of adenosine. Under optimized assay conditions, a linear range (1.0×10-8mol/L to1.0×10-7mol/L) is got with low detection limit of5.61nmol/L. The biosensor exhibits excellent selectivity and can provide a promising potential for aptamer-based adenosine detection (chapter7).(7) A time-gated fluorescence sensor for water content determination in organic solvents was proposed in this paper. A europium ternary complex (ETC) was synthesized and used as the fluorescence indicator in the fabrication of the fluorescence water sensor. To prevent leakage of the fluorophore, ETC was photo-copolymerized with acrylamide,(2-hydroxyethyl)methacrylate,2-hydroxy-2-methyl-1-phenyl-1-propanone, and triethylene glycol dimethacrylate on a glass surface treated with a silanizing agent. The time-gated fluorescence intensity of ETC decreased with increasing of water content in organic solvents. In the range of0.0%～8.0%(v/v), the time-gated fluorescence intensity of ETC changed as a linear function of water content. The detection limits were0.056%,0.042%, and0.033%for ethanol, tetrahydrofuran, and1,4-dioxane, respectively. The sensor exhibited satisfactory reproducibility, reversibility, and a short response time. The sensing membrane was found to have a lifetime of at least one mouth (chapter8). |
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