Quantum Dot @ Nanogel Ratiometric Fluorescent Sensor For Lead Ion Detection

Quantum dots（QDs）are a class of inorganic fluorophore with unique optical properties including high fluorescence quantum yield and anti-photobleaching ability,broadband absorption and narrowband emission.QDs are widely used for the development of fluorescent probes and sensors.Nanogel（NG）is a three-dimensional network structure formed by cross-linking polymers through physical or chemical methods on nanometer scale,showing the characteristics of adjustable structures,strong anti-fouling capability,and target enrichment.Encapsulation with the nanogel can improve the biocompatibility,physiochemical stability,detection sensitivity of sensors.Paper-based analysis devices（PADs）can be constructed by fixing sensors on a paper substrate.PADs have the advantages of low cost,simple operation,small sample consumption,and convenient carrying,finding high potential in the applications such as environmental/food safety on-site tests,and disease point-of-care tests.In this thesis,we prepared quantum dot micelle-spherical nucleic acid（QM-SNA）by coupling functional nucleic acids around a quantum dot micelle（QM）,and encapsulated multiple QM-SNA nanoparticles into a core-shell-type nanogel to construct a so-called QMSNA@NG fluorescent sensor for sensitive Pb²⁺ detection.Furthermore,by fixing the QM-SNA@NG fluorescent sensors on cellulose paper,PADs were developed for onsite detection of Pb²⁺ in real water samples.Featured with cascade DNAzyme walkingmediated signal amplification and ratiometric fluorescence signals,the PADs were demonstrated to provide a sensitive detection platform that is suitable for on-site and smart-phone assay of heavy metal ions.The main research contents include:1.Preparation of QM-SNA.By using one-step EDC/NHS condensation reaction,the QM-SNA was prepared by coupling a linker,NH2-PEG-Ad,and four kinds of functional nucleic acids including one aptazyme(Pb²⁺ aptamer hybridized with DNAzyme 1)and three quencher-modified substrates（substrate 1,2,3）on the surface of a quantum dot micelle.The as-prepared QM-SNA nanoparticles were characterized by infrared spectroscopy,tunneling electron microscopy（TEM）,and fluorescence spectroscopy.The results showed that multiple nucleic acid strands were coupled onto the QM surface,exhibiting a spherical structure,and the QM fluorescence was quenched.2.Target-triggered cascade DNAzyme walking-mediated signal amplification of QM-SNA.In the presence of target Pb²⁺,the aptamer unit of aptazyme specifically binds with Pb²⁺ to form G-quadruplex,and the DNAzyme 1 unit is released,which subsequently triggers cascade DNAzyme walking: DNAzyme 1 walks on the substrate 1 track,producing DNAzyme 2;DNAzyme 2 walks on the substrate 2 track,producing DNAzyme 3;DNAzyme 3 walks on the substrate 3 track,producing quencher-modified fragments;the three-level DNAzyme walking ends at the same time.The cascade DNAzyme walking drives the quenchers be separated from the QM,and the QM fluorescence is restored.The highly-efficient cascade DNAzyme walking of QM-SNA was investigated by total inner reflection fluorescence microscopy（TIRF）.3.Controllable construction of QM-SNA@NG fluorescent sensor for sensitive Pb²⁺ detection.With green QDs-contained aminated silica nanosphere as the core,the QM-SNA@NG was constructed in three steps: by using the condensation of EDC/NHS,the surface of silica nanosphere was modified with Poly（methyl vinyl ether-β-cyclodextrin）,P（MVE-β-CD）;by using the host-guest reaction between adamantane and β-cyclodextrin,the QM-SNA nanoparticles were anchored onto P（MVE-β-CD）;by using the EDC/NHS condensation reaction,NH2-PEG-NH2 was cross-linked with P（MVE-β-CD）to form the nanogel shell.The as-prepared QM-SNA@NG was characterized by TEM,confocal laser fluorescence microscopy,dynamic optical scattering measurement,and fluorescence spectroscopy,and the results showed that QM-SNA@NG has a core-shell structure,a particle size of ¹40 nm,electrical neutrality and dual emission.With the green QDs fluorescence as the reference signal,and the target concentration-dependent red QM fluorescence as the response signal,the QM-SNA@NG sensor realized quantitative Pb²⁺detection in ratiometric fluorescence.The achieved detection limit is as low as 0.5 pM,the linear dynamic range is 3 orders of magnitude.4.The fabrication of QM-SNA@NG paper analysis device for on-site and smartphone detection of Pb²⁺ in real water samples.The PAD was fabricated as follows: by the oxidative self-polymerization of dopamine,a polydopamine layer is formed on the surface of cellulose paper;by the Schiff base reaction between amino and aldehyde groups,NH2-PEG-Ad is coupled onto the cellulose paper;by the hostguest reaction between adamantane and β-cyclodextrin,QM-SNA@NG sensors are fixed onto the cellulose paper;the QM-SNA@NG-containing cellulose paper is cut,and the pieces are arrayed on a hydrophobic paper substrate.Under the irradiation of ultraviolet lamp,the PAD can be used for the quantification of Pb²⁺ with smart phone.By the spiking/recovery method,Pb²⁺ in real water samples were on-site assayed by the PAD,and the results were compared with the gold-standard IS-MASS method.