Establishment And Application Of Nanoparticle Encoded Microspheres Based Suspension Array

Inorganic nanoparticles possess unique function properties due to their special size effect,and meanwhile,polymeric microspheres are well-suited micro-carriers for biomedical applications due to their controllable size,large specific surface area and flexibility of functional group modification.Therefore,inorganic nanoparticles composited polymer microspheres can combine all those excellent properties derived from the both compositions,thus possess great potential for biomedical applications.Especially in recent years,a rising immunoassay technology called suspension array combining quantum dot encoded microspheres and flow cytometry have attracted much attention because it is a high-throughput multiplex detection platform with high sensitivity.In this work,we focused on nanoparticle encoded microspheres based suspension array,and work stated as follows was done:（1）Efficient preparation of magnetic/fluorescent polymeric microspheres by incorporating NIR-emitting CuInS₂/ZnS quantum dots and Fe₃O₄ magnetic nanoparticles into polymer via Membrane Emulsification-Emulsion Solvent Evaporation method.The as-prepared composite microspheres show excellent monodispersity,smooth surface with carboxyl group,uniform distribution of nanoparticle inside the microsphere.The microspheres also have excellent fluorescent property with high stability and environmental resistance,and good superparamagnetic for efficient immunomagnetic separation.Compared with Cd contained quantum dots used in the reported literatures,NIR emitting CuInS₂/ZnS quantum dots have advantages in biocompatibility and suppression of fluorescent quenching because of absorption of magnetic nanoparticles.A‘single wavelength’encoding model was successfully constructed to guide the encoding of NIR QDs with wide emission spectra.Then a‘single wavelength’encoding combined with size encoding was used to produce different optical codes by simply changing the wavelength and the intensity of the QDs as well as the size of the barcode microspheres.（2）Suspension arrays for multiplexing basing on the self-fabricated QD encoded microspheres and flow cytometric platform were established.The as-prepared NIR-emitting QD/MNP barcodes distinguished and detected five tumor markers in a single sample.Compared to the non-magnetic multiplex assay,the magnetic separation based multiplex assay showed enhanced sensitivity for all the tumor markers,which may have been attributed to the reduced non-specific biofouling.The multiplex detection based on NIR-emitting QD/MNP barcodes shows far more sensitive than ELISA.In order to validate the effectiveness of the magnetic QD barcodes for detecting serum spiked tumor markers,65 patient samples were simultaneously detected by using our magnetic QD barcode based 5-plex assay and clinical diagnostic methods including Electro-Chemiluminescence Immunoassay and Chemiluminescence Immunoassay.And the results show excellent performance of our assay when dealing with patient serum samples.These results indicate the promising application of NIR-emitting QD/MNP encoded microspheres for multiplex immunoassays.（3）Polyethylene glycol（PEG）grafted amphiphilic copolymers based QD encoded microspheres were prepared,and the as prepared PEG modified microspheres show obvious capability of non-specific biofouling suppression for enhanced detection sensitivity.Firstly,PEG were grafted onto poly（styrene-co-maleic acid）（PSMA）via esterification between carboxyl group of PSMA and hydroxyl group of methoxy polyethylene glycol（mPEG）.Grafting degree can be controlled by tuning the feed ration of PSMA and mPEG,and also PEG with different molecular weight were used.Then the as-prepared PEG-g-PSMA were dissolved in QD contained solutions for production of QD encoded PEG-g-PSMA microspheres via SPG membrane emulsification technique.The as-prepared microspheres were proved to be able to suppress nonspecific biofouling in two singleplex assays.The molecular weight and grafting degree of PEG are the two key factors for production of PEG microspheres with different effects of nonspecific biofouling suppression.In summary,our innovations are stated as follows:1)we are the first to prepare magnetic-fluorescent barcodes based on cadmium-free NIR-emitting QDs,the used NIR emitting CuInS₂/ZnS quantum dots have advantages in biocompatibility and suppression of fluorescent quenching because of absorption of magnetic nanoparticles,and the barcode based suspension assay were proved to have high sensitivity and clinical enhancement;2)A‘single wavelength’encoding model was constructed to guide the encoding of NIR QDs with wide emission spectra,thus improving the encoding capacity and application flexibility in suspension array of NIR QDs;3)Polyethylene glycol（PEG）grafted amphiphilic copolymers based QD encoded microspheres were prepared and proved to be capable in nonspecific suppression and sensitivity enhancement.