Establishment And Application Of Inorganic/Polymeric Microspheres Based Suspension Biochip System

Polymeric microspheres are well-suited carriers in biomedicalapplications for their large specific surface area, controllable size andcapability to be modified with various functional groups. In another hand,as one kind of important nano-materials, inorganic nanoparticles haveunique properties for their special size effect. Therefore, inorganicnanoparticles/polymeric composite microspheres can combine thesefunctional characteristics from both compositions, and possess greatpotential value in biomedical application fields. Especially, a biomedicalassay technology named suspension biochips, which was based onfluorescent microspheres, attracted much attention in recent years for itshigh-throughput and multiplex technical features. So in this work, wefocused on the establishment and application of inorganic/polymericcomposite microspheres based suspension biochip system, and thecontributions are as follows:(1) We achieved controllable fabrication of monodisperse carboxylcapped polymeric microspheres with various morphologies by integratingdispersion polymerization, seed polymerization, emulsion solvent evaporation approach and membrane emulsification technology. First,porous poly(St-DVB-MAA) microspheres were fabricated via seedpolymerization with monodisperse PS seeds synthesized by dispersionpolymerization. The porous structure and size of microspheres could beboth controlled by the properties of seeds and crosslinker concentrations.Meanwhile, the microspheres had carboxyl groups which come from theMAA added. Thereafter, we fabricated nonporous PSMA microsphereswith membrane emulsification-emulsion solvent evaporation method(MEESE method). Under proper emulsification conditions, the size ofPSMA microspheres was determined by the pore size of membrane, andthe surface anhydride groups of PSMA microspheres could be tranferredinto carboxyl groups via acid-catalyzed hydrolysis. Finally, hollowpoly(St-DVB-MAA) microspheres were synthesised via seedpolymerization with PS seeds fabricated in MEESE method. Themorphologies of these hollow microspheres could be controlled by themolecular weights of seeds.(2) According to the principle for fabricating polymeric microspheresand the structures of resultant polymeric microspheres, different methodswere used to fabricate inorganic/polymeric composite microspheres. First,a swelling-evaporation method (SE method) was developed to fabricateQD-microspheres with porous poly(St-DVB-MAA) microspheres, inwhich the swelling process was combined with gradual evaporation of thesolvent, thus gradual concentration of QDs in the dispersion solution. Thismethod was demonstrated to be an efficient method for improving thefluorescence intensity of resultant microspheres compared with the use ofswelling alone. Through this method, QD-microspheres encoded witheither single or multiple wavelength-emitting fluorescence spectra werefabricated effectively by adding different QDs into the swelling solution.Thereafter, to achieve a one-step fabrication, we developed an efficientMembrane Emulsification-Emulsion Solvent Evaporation (MEESE)method to fabricate the QD-microspheres. The QD-microspheres fabricated in this method showed excellent fluorescence stability in variousenvironments. And the fluorescence spectra of QD-microspheres could beconveniently controlled by changing the QDs in the dispersion phase.Finally, to further take the advantages of MEESE method, bi-functionalcomposite microspheres were successfully fabricated by adding magneticFe3O4nanoparticles and QDs into dispersion phase simultaneously.(3) Based on the self-fabricated QD-microspheres, we established asuspension biochip system on the flow cytometric platform. First, by thecombination of the advantages of MEESE approach in controllingQD-microsphere size and accurate encoding, a three dimensional barcodelibrary with the integration of signals in Forward Scatter (FS), FL1andFL4Channels was established in flow cytometer. Thereafter，the fiveindexes of hepatitis B viruses (HBV) were chosen as diagnosis targets，andthe detection experiments of HBV immunoassays were designed andcarried out in a flow cytometer. Based on the successful implementation ofsingleplex, cross-reaction and multiplex immunoassays of five HBV serummarkers, detection standard curves of HBV were established whichshowed good specificity and sensitivity. Then, in comparison withcommercial clinical detection technology, detections of human serumsamples were carried out. Finally, all the detection results verified that theself-established suspension biochip system was feasible and effective inthe biomedical assays. Beside these, by using self-fabricated magnetic andfluorescent microspheres, a specific magnetic separation for HBsAg wasachieved, which further promoted the applications of self-fabricatedcomposite microspheres in biomedical fields.In summary, the conclusions of innovations in our work are as follows:1) In the fabrication of QD-microspheres, a SE method was developed toimprove the fluorescence properties of QD-microspheres, and theadvantages of this method were further verified by comparison withconventional swelling method.2) The SPG membrane emulsificationtechnology was introduced into the fabrication of inorganic/polymeric composite microspheres. This method has unique advantages owing to itsmild fabrication conditions and high productivity. The resultant compositemicrospheres have good monodipersity, controllable size and excellentfluorescence properties.3) In the set-up of barcode library ofQD-microspheres, we take the advantage of membrane emulsification inthe size controlling of QD-microspheres, and the flow cytometric FS signal,which was correlative to the size of QD-microspheres, was introduced as anew encoding parameter for more barcodes. Finally, a QD-microspherebased suspension biochip system was firstly designed and applied to theimmunoassays of HBV successfully.