Study On Analytical Methods Of Silver Nanoparticles Enhanced Fluorescence And Their Applications

Fluorescence analysis is an important mean in biological analysis field.It is significant for development of biomedical science to improve the sensitivity of fluorescence analysis and accomplish the determination of extremely low content of biomarkers in clinical samples.Silver nanoparticles have excellent optical properties,and the fluorescence intensity of fluorescent dyes can be greatly enhanced when positioned near the surface of silver nanoparticles or films,which is called silver-enhanced fluorescence.This research work focus on silver-enhanced fluorescence and carry out a series of studies on the fluorescence enhancement,the mechanism and regularity of fluorescence enhancement,and develop highly sensitive and specific fluorescent methods for protein analysis.1.Fabrication of silver nanoparticle hybrid probes and fluorescence enhancement detection of IgEAn ultrasensitive protein assay method was developed based on silver nanoparticle(AgNP)hybrid probes and metal enhanced fluorescence.Two kinds of aptamer based silver nanoparticles,Aptamer/O1igomer-A/Cy3-modified AgNPs(Tag-A)and Aptamer/O1igomer-B/Cy3-modified AgNPs(Tag-B),were hybridized to form a silver nanoparticle aggregate,which could produce a red shift and broadening of the localized surface plasmon resonance(LSPR)peak.The enhanced fluorescence resulted from the increased content of Cy3 molecules and their emission resonance coupled to the broadened localized surface plasmon(LSP)of AgNP aggregates.The separation distance between Cy3 and AgNPs was 8 nm,which was the most optimal distance for metal enhanced fluorescence and the separation distance between adjacent AgNPs was about 16 nm.This was controlled by the lengths of the oligomer-A and the oligomer-B.The presented aggregate probes produced a wider linear range from 0.49 ng/mL to 1000 ng/mL with the detection limit reduced to 40 pg/mL(211 fM).2.Highly sensitive detection method for proteins based on the novel metal-enhanced silver nanostructureWe present a highly sensitive metal enhanced fluorescence(MEF)method based on a novel silver nanostructure fabricated with Cy5-functionalized silver nanoparticles(AgNPs)and AgNO3.The analytical performance has been demonstrated by microarray detection of streptavidin(SA)and human IgE.The fluorescence intensity can be enhanced substantially with the combined use of AgNPs and fluorescence enhanced solution(FES).Aptamers have been used for the preparation of Tag-C,which could accomplish IgE detection from 0.5 ng/mL to 16 ng/mL with the limit of detection is determined to be 0.25 ng/mL.SEM images show nanogaps exist in the aggregated silver nanoparticles and the nanogaps allow for the trap of fluorophores in the nanostructures that emit brighter light upon excitation.The silver nanostructures formed by Tags and FES proved to be an excellent platform for MEF of fluorophores whose excitation and emission occurred between 436 nm and 1000 nm.Finite-difference time-domain(FDTD)simulation has been carried out to confirm the enhanced electromagnetic field inside silver nanostructures,which could lead to strong overlap/resonance coupling and eventual fluorescence enhancement.3.Metal-enhanced fluorescence detection for proteins on antibody-conjugated silver plasmonic substrateThis part describes ultrasensitive fluorescent detection method through fabricating silver nanoshell substrate microarrays.Ag@Ag and Ag@Au core—shell nanoparticles with different size were first synthesized by seed-mediated growth method and the metal enhanced fluorescence of these nanoparticles on different fluorescent dyes are investigated.The results indicate Ag@Ag is a versatile and effective metal enhanced fluorescence material on different fluorophores while the enhanced fluorescence from Ag@Au was limited only to some of fluorophores with longer emission wavelength.When the Ag@Ag nanoparticles are functionalized with the aptamers and fluorescent dyes,a good analytical performance for simultaneous detection of human IgE and platelet-derived growth factor-BB(PDGF-BB)could be obtained.In this case,the detection limit for PDGF-BB was below 0.4 ng/mL.Furthermore,Ag@Ag was used to fabricate a plasmonic microarray substrate to further enhance the sensitivity of fluorescent detection.As a result,linear response to PDGF-BB concentration in the range of 16 pg/mL-50 ng/mL and detection limit is 3.2 pg/mL.In addition,the Ag@Ag modified plasmonic microarrays have still good recovery and no interface of human serum when PDGF-BB concentration was 2 ng/mL in normal human serum.The silver nanoshell plasmonic substrates show their high specificity and sensitivity for protein microarray detection.4.Silver nanoparticle-enhanced fluorescence resonance energy transfer sensor for human PDGF-BB detectionA silver nanoparticle(AgNP)-enhanced fluorescence resonance energy transfer(FRET)sensing system is designed for the sensitive detection of human platelet-derived growth factor-BB(PDGF-BB).Fluorophores-functionalized aptamers and quencher-carrying strands hybridized in duplex are coupled with streptavidin(SA)-functionalized nanoparticles to form AgNP-enhanced FRET sensor.The resulting sensor shows lower background fluorescence intensity in the duplex state due to the FRET effect between fluorophores and quenchers.Upon the addition of PDGF-BB,the quencher-carrying strands(BHQ-2)of the duplex are displaced leading to the disruption of the FRET effect.As a result the fluorescent intensity of the fluorophore-aptamer within the proximity of the AgNP is increased.When compared to the gold nanoparticle(AuNP)based FRET and bare FRET sensors,the AgNP-based FRET sensor showed remarkable increase in fluorescence intensity,target specificity and sensitivity.Results also show versatility of the AgNP in the enhancement of sensitivity and selectivity of the FRET sensor.In addition,a good linear response was obtained when the PDGF-BB concentrations are in the range of 6.2 ng/mL-50 ng/mL and 100 ng/mL-500 ng/mL with the detection limit is of 0.8 ng/mL.5.Ultrasensitive and fast fluorescent bioassay based on fluorescence enhancement of silver nanoparticlesAn ultrasensitive,fast and specific fluorescent platform for protein detection was developed.In this protocol,silver nanoparticles were conjugated with paramagnetic particles(MPs-Ag)for target capture,concentration and separation,fluorescent dyes functionalized silver nanoparticles(Tag)for generating signals.The presented method was highly sensitive and specific with a detection limit of 2.2 pM for thrombin,and no significant interference was observed for other proteins such as human serum albumin(HSA),lysozyme and IgG.This novel approach of combining the magnetic separation and concentration of MPs-Ag,aptamer recognition and fluorescence enhancement of Tag,can be successfully used to enhance the sensitivity of detecting ultra-low levels of target proteins or biomolecules.