Preparation And Application Of Fluorescence-Encoded Microspheres Based On Hydrophobic Conjugated Polymer-Dye Combination

Suspension array technology(SAT)based on spectrally or optically encoded microspheres is suitable for simultaneous and fast detection of multiple analytes in one sample with small volume.It has been used for immunoassay,nucleic acid detection,etc.;and demonstrated great potential as a general analytical platform in the field of clinical diagnostics.The type,quantity,performance and price of encoded microspheres influence the development of the technology.Therefore,it is of great practical significance to develop more fluorescent encoded microspheres with simple preparation,low cost and good performance.From the perspective of practical application,we proposed a strategy for preparing binary encoded microspheres array based on hydrophobic conjugated polymer and dye combination,and explored the decoding of the array and biomolecule detection based on these spheres.The conjugated polymer poly(p-phenyleneethylene)(PPE)and the organic dye Nile Red(NR)were used as two different type of fluorophores,and monodispersed amino-modified porous polymeric spheres,poly(glycidylmethacrylate)microspheres(APGMA)were used as substrate spheres.Loading the fluorophores sequentially onto the substrate spheres via adsorption by immersing the spheres in the dipping solution of fluorophores resulted the APGMA-PPE-NR spheres.After optimization,eight sets of binary encoded microspheres having eight different emission intensities and two different emission wavelengths were prepared.These microspheres exhibited a variety of colors observed by laser confocal scanning microscopy,and the color of these microspheres in the macroscopic state under natural light and UV light were consistent with the signals obtained from the fluorescence emission spectra.In addition,these microspheres were found to have good stability against washing,photobleaching and thermal treatment.According to the encoding strategy of the wavelength-intensity combination,the concentrations of the two fluorophores in the dipping solution were changed to prepare an array of 64-code APGMA-PPE-NR spheres.It was verified by flow cytometry that the fluorescence signals of these microspheres could be decoded individually,and the resolution identification of some mixed samples was realized,indicating that this differential coding is likely to be recognized in practical applications.In addition,a sandwich immunoassay for detection of goat IgG was performed,and the feasibility of the resultant encoded microspheres for suspension arrays was demonstrated.