Preparation Of Fluorescence-Encoded Microspheres And Construction Of Fluorescence-Anticounterfeiting System Based On Energy Transfer

Fluorescence energy transfer,as a fluorescence phenomenon produced under specific conditions,has been widely used in many fields.In recent years,researchers have begun to explore the preparation of fluorescent microspheres,as the key materials for flow cytometry,and the construction of fluorescence-anticounterfeiting systems based on energy transfer.At present,most methods for coding fluorescent microspheres reported in the literature are based on the linear relationship between the emission intensity and the concentration of the fluorophores.However,linear coding is susceptible to many factors,which may limit the number of fluorescence-encoded microspheres.Non-linear encoding based on energy transfer may circumvent these effects and achieve a larger amount of coding.However,there are very few relevant literatures about the coding of fluorescent microspheres based on energy transfer;even fewer reported in-depth study;in addition,the resulted fluorescence-encoded microspheres are also difficult to meet the increasing practical application requirements.On the other hand,counterfeits and shoddy products have become a global problem,and anticounterfeiting is an effective means to suppress this issue.However,most of the existing anti-counterfeiting methods have the disadvantages of simple encryption,easy imitation,complicated operation during anticounterfeiting identification,and high requirements on instruments.The anticounterfeiting method by adjusting the color of the fluorescent patterns based on energy transfer reveals many advantages.However,there are only few studies about it,thus more research to provide effective anticounterfeiting encryption strategy is necessary.Based on the principle of fluorescence energy transfer,this thesis designs a strategy for coding fluorescent microspheres and provides a method for applying energy transfer to anticounterfeiting encryption.The specific contents are described as follows.Before coding fluorescent microspheres,the preparation and characterizations of fluorescent microspheres were explored on the basis of our previous work.The studies were mainly about the influence of outer-layer micelles on the bioconjugation capability of fluorescent microspheres and the explanation of the principle and operations of fluorescence compensation for flow cytometry.First,introducing of fluorescent conjugated polymer poly(p-phenylenevinylene)(PPV)as the signal source and micelles(PS Am)as the interlayer spacers of PPV onto the substrate spheres,it confirmed the effectiveness of using micelles as the interlayer spacers of fluorescent conjugated polymers to prepare microspheres with strong emission signals;at the same time,rhodamine dye(RBITC)-labeled goat anti-human antibody(IgG-RBITC),exhibiting different fluorescence signal from microspheres,was chosen as the biomolecule representative to combine with the microspheres.Consequently,the emission spectra confirmed that micelles effectively provided biomolecule binding sites for microspheres.Secondly,based on the in-depth analysis of previous work,that is,the process of characterization of the bioconjugation capability of two APGMA-CP microspheres by introducing two poly(p-phenyleneethylene)(PPE)polymers onto substrate microspheres,using bovine serum protein labeled with FITC(BSA-FITC)as biomolecule representative,systematically elaborated the specific principle and operations of fluorescence compensation for flow cytometry.The theory and practical basis are provided for effectively deducting interference of signals based on flow cytometry characterization.Then energy transfer was applied to the preparation of fluorescence-encoded microspheres,and the strategy of "non-linear encoding" of fluorescent microspheres based on energy transfer was studied in depth.We proposed two systems.In the first system,the coding based on energy transfer between two coding elements on the surface of microspheres was explored.PPV nanoparticles(PPV NPs)as energy donor and rhodamine 6G(R6G)as energy acceptor,were introduced onto the surface of substrate microspheres(SPSDVB);by regulating the energy transfer between two coding elements,9 kinds of fluorescence-encoded microspheres(S-PPRn)were obtained.The second system further explored the energy transfer of two coding elements mixed into the substrate microspheres.Carbon dots(C-Dots)as energy donor and R6G as energy acceptor were co-introduced into the pores of substrate microspheres(SPSDVB);by regulating the energy transfer between the them,9 kinds of fluorescence-encoded microspheres(S/C-Dots/R6Gn)were obtained.In order to prevent the leakage of coding elements,a melamine-formaldehyde resin layer(MF)was prepared on the surface of the above fluorescence-encoded microspheres.Under the protection of the MF layer,these microspheres can keep stable emission signals in different solvent environments.Moreover,the preparation process of MF was found to have negligible influence on the encoded signals.Afterwards,the microspheres were modified by Silanization reaction to provide biomolecule binding sites on the surface of these fluorescent microspheres.Finally,the goal of characterizing the signals of these fluorescence-encoded microspheres using flow cytometry was achieved.Finally,a fluorescence-anticounterfeiting system based on energy transfer was constructed.The strategy of anticounterfeiting encryption based on ternary energy transfer and anticounterfeiting identification using the excitation-wavelength-dependent of C-Dots materials were explored.C-Dots,FITC and R6G aqueous solutions were prepared as blue,green and red fluorescent inks respectively.Through hand-painting anticounterfeiting patterns,it was verified that the color change of anticounterfeiting patterns during identification process was resulted from the synergistic effect of changes in absorption intensity and the degree of ternary energy transfer.Then,ink-jet printer was used to precisely control the proportion of fluorescent inks,and the printing of anticounterfeiting patterns on A4 paper and Chinese paper money was realized.The anticounterfeiting identification can be performed by switching the lights,and the second-level anticounterfeiting identification was initially achieved.In all,a concept of anticounterfeiting strategy based on the combination of energy transfer and excitation-wavelength-dependent of the C-Dots to adjust the emission color of patterns was realized.In summary,based on the energy transfer principle,this thesis proposed and realized the strategy for preparation fluorescence-encoded microspheres and constructed and developed the strategy for fluorescence-anticounterfeiting.This study not only extended the application of energy transfer theory,but also help the widespread of flow cytometry with fluorescent microspheres as the signal carriers,as well as promote the development of the safe and effective fluorescent anticounterfeiting technology.