Synthesis of Magnetic Fluorescent Bead and Its Applications in Fluorescent Immunochromatographic Assay

Author:Guo Liang

Supervisor:xiong yong hua


Degree Year:2019





Bifunctional magnetic fluorescent beads(MFBs)exhibit both superparamagnetism and fluorescence.Biological recognition element functionalized MFB has been widely used in biomedical bimodal imaging attributed to its ability to combine with target specifically,and to make it exhibit both fluorescent and magnetic signal.Owing to the preconcentration/purification via imuno-magnetic separation,the MFB-based analytical methods could markedly improve the sensitivity and accuracy for trace target analytes in complex samples.Circulating tumor cells(CTCs)have been listed as a biomarker for early diagnosis of tumor(metastasis)and prognosis.However,CTCs are extremely rare in peripheral blood of metastatic patients and blood matrix may interfere with their detection.Therefore,the detection process needs a magnetic bead-based separation enrichment step refers to isolation of CTCs from the blood.Herein,a facile one-step method for direct self-assemble of oppositely charged carboxyl quantum dots(QD)onto the surface of amine modified magnetic silica bead was developed to prepare bifunctional magnetic fluorescent beads.The ethanol content,pH value and the dosage of QD for self-assembly were optimized to regulate the assembly efficiency and avoid aggregation of resultant MFBs.Under optimal condition,two kinds of hydrophilic core-shell structured bifunctional magnetic fluorescent beads with diameters of 180 nm were prepared,which exhibited relative strong fluorescence with different emission peak at 590 nm and 630 nm,respectively.Then,the streptavidin modified 180 nm MFB and biotinylated anti-EpCAM antibody were used for magnetic separation.And the separated MCF-7 cells were detected using fluorescence microscope.Immunochromatographic assay(ICA)is a widely used point-of-care test technology in environmental monitoring,food analysis,and in vitro diagnosis because of its simplicity,rapidity,portability,cost-effectiveness,and user-friendliness.However,its application for the ultra-trace analyte detection in complex samples remains challenging.The matrix interferences of complex samples and insufficient analytes are major factors decreasing the sensitivity of ICA.Specific preconcentration of analytes based on immunomagnetic separation(IMS)is a simple and feasible strategy to overcome these challenges and improve the detection sensitivity of ICA.In the present study,we prepared a novel core/shell structured bifunctional MFBs with high fluorescence intensity and good magnetic responsibility via a facile one-pot ultrasonic emulsification.Then IMS-based fluorescent competitive ICA platform for detection of small molecules and fluorescent sandwich ICA platform for detection of macromolecules were established using the resultant MFBs as reporters,and applied in detection of trace aflatoxin B1(AFB1)in dark soy sauce and human immunodeficiency virus(HIV p24 antigen)in human serum.The MFB used in this study were fabricated via a facile one-pot ultrasonic emulsification by encapsulating numerous octadecylamine-coated CdSe/ZnS QDs(OC-QDs)and oleic acid-modified iron oxide nanoparticles(OA-IONPs)into poly(methyl methacrylate)(PMMA)and poly(maleic anhydride-alt-1-octadecene)(PMAO)composites.The as-prepared MFBs showed a distinct core/shell structure,where QDs and IONPs were mainly distributed in the outer layer of MFBs,leading to high fluorescence intensity and good magnetic responsibility.Moreover,ultrasonic emulsification with different ultrasonic power can be used to prepare MFBs conveniently with tunable sizes ranging from nano-level to micron-level as reporters of ICA.As a result,the MFBs with diameters of 180 nm and 250 nm used in fluorescent competitive ICA platform and sandwich ICA platform were prepared via ultrasonic emulsification with ultrasonic powers of 104.5 W and 85.5 W,respectively.The saturation magnetization of these two MFBs were 18.2 and 14.1 emu/g,and were45.4%and 35.3%that of the OA-IONPs,which was much higher than the mass percent of IONPs doped in MFBs(11.8%).Meanwhile,these two MFBs exhibited226 times and 367 times brighter luminescence than the corresponding QDs,leading higher output fluorescent signal to enhance ICA performence.The resultant MFBs of 180 nm were then used as a reporter of fluorescent competitive ICA for the detection of trace aflatoxin B1(AFB1)in dark soy sauce.To improve the sensitivity and accuracy of ICA,we used anti-AFB1 antibody-labeled MFBs to enrich AFB1 molecules and eleminate matrix interferences by removing the interfering components in dark soy sauce samples.Under optimal detection conditions,the MFB-based ICA(MFB-ICA)exhibited a dynamic linear detection of AFB1 in sauce extract over the range of 5-150 pg/mL with a half maximal inhibitory concentration of 27.29 pg/mL(n=3).The detection limits for AFB1 in sauce extract and real dark soy sauce were 2.84 and 48.69 pg/mL,respectively,which are considerably better than those of previously reported fluorescent bead-based ICA methods.The analytical performance of the proposed MFB-ICA in terms of selectivity and accuracy was investigated by analyzing AFB1-spiked dark soy sauce samples.The developed MFB-ICA exhibits a certain extent of cross-reactivity(CR)with AFG1,and negligible CR with other six mycotoxins.The average recoveries for the intra-assay ranged from 97.24%to 102.76%,with a coefficient of variation ranging from 0.63%to 5.10%.The results for the inter-assay ranged from 92.91%to103.44%and 2.95%to 7.3%,respectively.The reliability of the proposed method was further confirmed by ultra-performance liquid chromatography with fluorescence detection.The anti-HIV p24 antibody functionalized MFBs of 250 nm were used as reporters of fluorescent sandwich ICA for the detection of HIV p24 antigen in human serum.The results of detection of HIV p24 in spiked PBS buffer showed that the lowest limit of linear detection range using traditional ICA is 8.16 times of the lowest limit using IMS-based ICA,which verified effect of IMS to improve the sensitivity of ICA.Under optimal detection conditions,the MFB-based ICA(MFB-ICA)exhibited a dynamic linear detection of HIV p24 in human serum over a wide range of 0.244–1000 ng/mL with the LOD of 0.16 ng/mL.Meanwhile,the MFB-ICA exhibits negligible cross-reactivity with common proteins in serum,including CRP,PCT,PSA,CEA and AFP.The average recoveries for the intra-assay ranged from 87.53%to104.03%,with a coefficient of variation ranging from 0.45%to 1.94%.The results for the inter-assay ranged from 95.35%to 111.57%and 6.18%to 14.94%,respectively.