Biosensors Based On Quantum Dots For Biological Analyses

Quantum dots(QDs),possessing unique optical properties including high fluorescence quantum yield,broad-band absorption,size-dependent narrow-band emission,and resistance to photobleaching,have emerged as a new class of fluorophores.Fluorescent biosensors based on QDs have significant advantages such as high sensitivity,good selectivity,simple and low cost,promoting their applications in biomedical research.In this thesis,multi-colored and multi-structured QDs were synthesized and further functionalized by means of bioconjugation.On this basis,DNA microarray,ratiometric sensor and QD-encoded microbeads were designed for single nucleotide polymorphism analysis,intracellular Pb2+ detection and multiplexed detection of microRNAs,respectively.The main contents are as follows:1.Multi-colored and multi-structured QDs were synthesized.Organic soluble CdSe/ZnS and CdSe/CdS/ZnS core/shell QDs were synthesized following the "successive ion layer adsorption and reaction(SILAR)" method.Organic soluble alloying CdxSe1-xZnyS1-y,ZnCdSe and ZnCdS/ZnS QDs were synthesized according to the "one pot" method.The as prepared QDs exhibit high quantum efficiency,narrow full width at half maximum(FWHM),and size(composition-)-tunable emission.2.Via bioconjugation,streptavidin-coated QD(strAV-QD)was prepared and successively applied in molecular beacon(MB)microarray assays by using the strAV-QD to label the immobilized MB,avoiding target labeling and meanwhile obviating the use of amplification.The MBs are stem-loop structured oligodeoxynucleotides,modified with a thiol and a biotin at two terminals of the stem.With the strAV-QD labeling an "opened"MB rather than a "closed" MB via streptavidin-biotin reaction,a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray,with a signal-to-background ratio of 8.The immobilized MBs can be perfectly regenerated,allowing the reuse of the microarray.The MB microarray also is able to detect single nucleotide polymorphisms,exhibiting genotype-dependent fluorescence signals.It is demonstrated that the MB microarray can perform as a 4-to-2 encoder,compressing the genotype information into two outputs.3.Also via bioconjugation,Pb2+ aptamer modified red-emitting QDs(apt-rQD)were prepared.A dual-emision fluorescent sensor was successively constructed by assembling the apt-rQD onto the surface of silica-coated green-emitting QDs(gQD/SiO2)-labeled graphene oxide(GO).For this sensor,rQD is the fluorophore;the aptamer is the recognition element for Pb2+;gQD/SiO2 is the reference for fluorescent evolution.Graphene oxide(GO)functions as the quencher,which quenches the fluorescence of InP/ZnS QDs through nano-metal surface energy transfer(NSET)mechanism.Upon binding with Pb2+,the aptamer suffers from a conformation change from single-strand to G-quadruplex,which increases the distance between fluorophore and GO.As a result,the fluorescence of rQD recovers while the fluorescence of gQD@SiO2 remains unchanged.Variations of the dual-emission intensity ratios display continuous color changes from green to orange by increasing the concentration of Pb2+,which can be clearly observed by the naked eye.Since the apt-rQD,GO and gQD/SiO2 exhibit no cytotoxicity,the ratiometric sensor is highly compatible for detecting intracellular Pb2+.4.A protocol of direct implementation of hybridization chain reaction(HCR)on silica encapsulated quantum dot-encoded microbeads(Qbead@SiO2)was proposed for colorimetric detection of miRNAs.Encapsulation of 100 nm-thick silica shell confers Qbeads with remarkably improved chemical-and photostability,allowing efficient bioconjugation,target binding and HCR on the surface.Simply by the presence of two hairpin species,the Qbead@SiO2—HCR assay remains cost-effective,but significantly improves the detection sensitivity compared to existing Qbeads-based assays.A dynamic range of 4 logs,and a detection limit of 700 zmol are achieved,owing to HCR-induced fluorescence signal amplification.More importantly,the HCR on Qbead@SiO2 results in color difference amplification which for the first time,allows color analysis be a conceivable quantification method for the Qbeads-based assay.With benefits of HCR-induced color difference amplification,four miRNAs extracted from human breast cancer cells can readily be quantified by distinct emission-color changes of four barcodes.The Qbead@SiO2—HCR assay with color analysis could potentially enable the development of point-of-care diagnostics platforms.