Quantum Dot @Nanogel Sensor And Paper-based Analysis Device For Multiplex Detection Of Heavy Metal Ions

Heavy metal ion pollution has caused serious harm to human health.Therefore,it is of great significance to monitor the heavy metal ions in various water environments.Functional nucleic acids such as aptamers,DNAzymes and aptazymes,featuring remarkable binding ability toward targets,can be combined with various nanoparticles to construct nano-biosensors.Nanogel（NG）is a three-dimensional porous network on the nanometer scale,characteristic of high water content,strong anti-fouling capability and good target-enriching ability,providing a biocompatible carrier for nano-biosensors.Paper-based analysis devices（PADs）that are made by fixing sensors on paper base,are advantageous in terms of low cost,good flexibility and portability.The PADs have become powerful tools for on-site environment monitoring,medical point-of-care test and other fields.In this thesis,we have developed a spherical nucleic acid-structured nano-biosensor,combining with NG nanocarrier and nanoparticle-doped photonic crystal-structured paper base,for highly sensitive detection of heavy metal ions.Based on aptazyme walker-mediated signal amplification of SNA,target enrichment of nanogel and local surface plasma enhancement of plasma photonic crystal substrate,the nano-biosensors/PADs can be used for smart phone detection of heavy metal ions,by coupling with a specifically-designed detection box.The main research contents are as follows:1.Controllable preparation of multicolor quantum-dot micelle spherical nucleic acids（QD-SNAs）.First,green-,yellow-and red-emission water-soluble QDs,respectively,were obtained via membrane hydration reaction;Second,multicolor QD-SNAs,respectively,were prepared by coupling aptazyme and BHQ-modified substrate（hairpins）on the QD.The physicochemical characterizations show that the QD-SNAs all have a spherical structure,and the fluorescence is quenched.2.Target-triggered aptazyme（bipedal walker）-mediated signal amplification of QD-SNA.The aptazyme was specifically designed to have a“Zn²⁺DNAzyme-aptamer-Zn²⁺DNAzyme”structure.In the absence of target ion,the aptazyme was hybridized with a DNA sequence to form a triple-helix where the dual Zn²⁺DNAzyme’s catalytic activities were deactivated.In the presence of target ion,the aptamer favorably binds with the target,driving the aptazyme to be separated from the QD-SNA,and simultaneously the dual Zn²⁺DNAzyme’s catalytic activities were activated.The dual Zn²⁺DNAzyme can shear the neighboring substrates（hairpins）and drive the aptazyme to walk autonomously in a bipedal manner,leading to the separation of BHQ from the QD-SNA and the recovery of QD fluorescence.The specificity of target recognition was verified by gel electrophoresis and circular dichroism.The kinetics of bipedal walker on QD-SNA was monitored by total internal reflection inverted fluorescence microscopy（TIRF）,and the walking time was determined to be 30 min.3.Controllable construction of core-shell QD-SNA@NG sensors and their use for detection of heavy metal ions.The QD-SNA@NG sensors were constructed by three steps:The amino silica spheres were linked with polymethyl vinyl ether-β-cyclodextrin（P（MVE-β-CD））by using EDC/NHS reaction;Adamantane（Ad）-anchored QD-SNA was connected to P（MVE-β-CD）via host-guest reaction;The P（MVE-β-CD）was further crosslinked with diamino polyethylene glycol（NH₂-PEG-NH₂）via EDC/NHS condensation reaction.The physicochemical characterizations show that the nano-biosensors have a“silica core-NG shell”structure with～110 nm in size,and are electrically neutral.Based on the signal amplification of QD-SNA and target enrichment of nanogel,the QD-SNA@NG sensors show high detection sensitivity towards Pb²⁺,Cd²⁺and Hg²⁺,with detection limits（3σ）of～1 p M and linear dynamic range of～3 logs.4.Construction of microfluidic PADs and their use for smart phone detection.First,with gold nanoparticles（Au NPs）-modified silica spheres as template,plasma-photonic crystal-structured nitrocellulose substrate was prepared by template deposition,carboxylated nitrocellulose filling and hydrofluoric acid etching.The obtained substrate was modified with adamantane polyethylene glycol（Ad-PEG-NH₂）via EDC/NHS condensation reaction and subsequently linked with QD-SNA@NG sensors via host-guest reaction.Second,by using the batik technology,the hydrophilic and hydrophobic areas were printed on a sheet of cellulose paper.Finally,the PADs were fabricated by stacking the QD-SNA@NG sensors-nitrocellulose substrates on the detection zone of patterned cellulose paper.The plasma-photonic crystal-structured nitrocellulose substrate showed a remarkable fluorescence enhancement,attributed to slow light effect of photonic crystal and local surface plasmon resonance of Au NPs.As such,the PADs can realize highly sensitive detection of Pb²⁺,Cd²⁺and Hg²⁺with a smart phone.The detection limits（3σ）were calculated to be～10 f M,and the linear dynamic range was defined to be～5 logs.By spiking/recovery experiments,the PADs were demonstrated to be capable of on-site smart phone detection of heavy metal ions in real water samples.