Fluorescence Sensor Chip Based On Optical Tweezers And Upconversion Luminescence Nanolabeling And Its Applications In Biological Analysis

The detection of pathogens,viral nucleic acids,tumor-related markers and other biomolecules have important significance for the determination of disease types and monitoring of disease treatment.Therefore,the development of reliable,sensitive and fast detection methods with high throughput is becoming a hot topic.Optical sensors based on upconversion luminescence have unique advantages in the detection of biological molecules,such as low background fluorescence,controllable sizes and shapes,and multi-color emission.However,common fluorescence imaging systems and flow detection platforms such as flow cytometer are often not equipped with near-infrared excitation light sources.Therefore,the development of imaging analytical techniques or microfluidics-based biosensors in combination with optical sensing strategies based on upconversion luminescence for biomolecule detection are restricted.The work of this thesis is based on the laboratory-built optical tweezers imaging analysis platform and home-constructed microfluidic flow detection platform.We combine upconversion luminescence with some sensing strategies,such as luminescence resonance energy transfer(LRET)effect,DNA molecular machine and indirect immunofluorescent labeling,to construct different strategies for detection of biomolecules.The main contents of this thesis are as follows:(1)We propose a novel analytical strategy by incorporating upconversion LRET,optical tweezers and DNA walking amplifier into the bead-supported imaging analysis,and apply it to detect mi RNA-21 sequences relating to a potential cancer biomarker for breast tumor.Specifically,luminescence confined UCNPs with a sandwhich structure(SUCNPs)are first prepared and then modified with PAA molecules by ligand exchange method.After assembling with ASBs,DNA walkers and DNA substrates are co-conjuated onto the surface SUCNPs.Before identifying the target mi RNA sequences,the walkers are locked by locked DNA and the upconversion luminescence is markedly quenched by energy acceptors(BHQ-1)through LRET effect.In the presence of mi RNA-21 sequences,the walkers are released by reason of strand displacement mechanism.Finally,the walking behavior is driven by the cleavage of nicking endonuclease and the recovery luminescence signals determined as gray-level are measured with optical tweezers platform equipped with EMCCD by optical trapping of a single target bead.The proposed array has ultra-high sensitivity and a favorable specificity for discriminating single base mismatching,and the recovery ratio is linearly related to the logarithm of[mi RNA-21]ranging from 100a M to 3 p M with a detection limit down to 69 a M.More importantly,the proposed analytical approach can be applied to accurately detect the level of mi RNA-21sequences in as few as 5 MCF cells,which presents response capability for tracing extremely low abundance targets even from single cell level.More importantly,this approach is used to accurately detect free targets in human serums and venous blood samples from liver breast cancer patients,exhibiting a good application prospect in bioassay and clinical diagnosis,and revealing a great clinical diagnosis prospect.(2)We construct microfluidic chip assisted flow platform by integrating upconversion luminescence nanolabeling with fluid focusing strategy,and apply it to detect E.coli O157:H7.Specifically,OA-capped Na YF₄:Yb,Er upconversion luminescence nanoparticles(OA-UCNPs)are first prepared using the thermal decomposition method and then are modified with PAA molecules by ligand exchange method.SPA is immobilized at the surface of UCNPs by amidation reaction.Anti-E.coli O157:H7 polyclonal antibody is used as recognized molecule to recognized preliminarily,then antibody binding Staphylococal Protein A(SPA)labeled with UCNPs is used to provide luminescence signals.The luminescence signals from target bacterias in chip are measured with microfluidic flow platform equipped with a 980nm laser by fluid focusing single target bacterium,and recorded by the self-designed software.Finally,the proposed flow analytical approach is applied to preliminarily detect E.coli O157:H7.