The Study On Protein Microarray Detection Method Based On Fluorescence Nanoparticle Probes

Fluorescence analysis is one of the most common methods in biology detection,which is widely used for a variety of bioanalytical purposes,such as detection of DNA,protein and other biomolecules,cellular labeling and in vivo imaging.With the deepening research of life science,it is more and more important to search for fluorescence analysis methods for high throughput detection and new type of sensors.In recent years,the applications of nanomaterials in the fluorescence analysis improved performance of fluorescent probes in biology analysis greatly.Study and preparation of nanomaterials having fluorescent activity and apply them in the biology analysis are of great significance in the rapid development of life science.In this work,we research and prepare fluorescent probes of silver nanoparticles and core shell silica nanoparticles doped with dyes,and apply them to the high sensitive detection on aptasensors and protein microarrays.The main content includes the following aspects:1.A novel aptasensor based on silver nanoparticle enhanced fluorescenceIn the present study,we design a novel aptasensor based on silver nanoparticle enhanced fluorescence for the detection of adenosine.First,the distance dependence nature of silver nanoparticle enhanced fluorescence was investigated through fluorescent dyes modified oligonucleotides to control the spacing distance between dyes and AgNP.The results showed that the fluorescence intensity reached the maximum value with the spacing distance of dyes about 8 nm from AgNP surface.The fluorescence intensity decreases when the spacing distance is either above or below this value.Based on this result,a fluorescence switch is constructed.In the"OFF" state,without the target molecules,there is a greater spacing distance between the Cy3 dyes and the AgNP giving comparatively lower fluorescence intensity.While in the "ON" state,in the presence of target molecules,the fluorescence signals increased for the conformation structure change of the aptamer which shorten the spacing distance between the Cy3 dyes and the AgNP to 8 nm.Using adenosine as the target,the aptasensor produced a linear range from 200 nM to 200 ?M and the detection limit was 48 nM.The aptasensor was also found to be specific in targeting adenosine.The presented method shows a new strategy of combining aptamer recognition and silver nanoparticle for fluorescence signal enhancement and increasing sensitivity.2.Aptamer-Based Silver Nanosensor for Multiple Protein DetectionA novel aptamer-based silver nanosensor was prepared for one-spot simultaneously detection of multiple proteins.ss-DNA modified AgNPs were immobilized on the aldehyde coated glass slide to form AgNP array.Then dyes labeled aptamer sequences were allowed to hybridize with their complementarystrands assembled on the surface of AgNPs.The target proteins were introduced to associate with the corresponding aptamers to form the aptamer/target complexes.The removal of the aptamer/target complexes resulted in the remarkable decrease of fluorescent signals.This nanosensor is found to be highly sensitive for the detection of protein.When thrombin was employed as a sample model,the limit of detection of optimized nanosenor was 0.4 fmol with linear response of 0.8 fmol to 0.5 pmol.We further demonstrated the multiple proteins detection of IgE and thrombin using multicolor silver nanoprobes,which shows effective recognition of the relative protein individually or simultaneously.This silver nanosensor offers a uniquely heterogeneous approach for protein detection with several advantages such as high sensitivity,rapidity,high throughput,and miniaturization.3.Aptamer silver nanoparticle fluorescent probe for the sandwich immunoassay on protein microarrayAptamer silver nanoparticle probes were prepared and applied for the sandwich immunoassay fluorescence detection on protein microarray to achieve the high sensitive detection of single protein and multiplex detection.We first use it for the detection of thrombin.Because of the enhanced fluorescence of silver nanoparticles and large surface area of nanoparticle,this protein capture probe is very sensitive.The detection limit of this method is 6.2 pM,which is 80 times lower than the method using aptamers directly.And PDGF-BB was also detected using this probe,the detection limit decreased by 8 times comparing to use aptamers directly.In order to accomplish multiplex detection of proteins,we use multicolor silver probes to capture thrombin and PDGF-BB simultaneously in the sandwich immunoassay.And the capture ability of this probe for mixture proteins is further researched.This new method of sandwich immunoassay has great potential in the fluorescence detection of protein microarray.4.Rhodamine B doped silica nanoparticle labels for protein microarray detectionA core-shell Rhodamine B-doped SiO2 nanoparticle was synthesized and its fluorescent intensity was found to be 1000 times higher than that of individual Rhodamine B molecule.The doped nanoparticles were further conjugated with streptavidin and the resulting nanoparticles were used in the detection of reverse-phase protein microarrays,in which human IgG of various concentrations was first immobilized on aldehyde-modified glass slides and then biotinlyated goat anti human IgG as well as the labeled nanoparticles were sequentially conjugated.The calibration curve is linear over the range from 800 fg to 500 pg and the limit of detection is 100 fg,which is 8 times lower than that of streptavidin-labeled Cy3 fluorescent dyes.The dye-doped SiO2 nanoparticles show potentials for the protein array detection.