Application Of Quantum Dot Nanobeads In Immunochromatographic Assay

Immunochromatographic assay?ICA?has been widely applied in clinical diagnosis,food safety,and environment monitoring owing to its advantages of rapidity,user friendliness,low cost,and simplicity.However,the conventional ICA commonly suffers from the relatively low sensitivity owing to the low brightness of traditional 20?30 nm gold nanoparticles?GNPs?,and thereby constraining its further application for the trace analytes.Therefore,it is urgently required for developing some novel strategies to improve the sensitivity of ICA.Compared with the traditional GNPs,fluorescent labels can dramatically enhance the sensitivity of ICA because of their remarkable optical intensity.Among currently reported fluorescent labels,quantum dots?QDs?have widely used to develop the high sensitive and/or multiplexed ICAs owing to their superior advantages including strong luminescence,broad excitation with narrow fluorescent emission spectra,and large stokes shift.Encapsulating numerous QDs doped into polymer matrix can obtain dramatically enhanced brightness and stability.Therefore,QDs beads?QBs?could be a promising label to further improve the sensitivity and stability of ICAs..In this thesis,the relevant applications of QBs in ICAs are listed as follows:1)QBs were prepared by encapsulating numerous CdSe/ZnS QDs into polymer matrix via a simple ultrasonic emulsification-solvent evaporation?UESE?method.The resultant QBs showed good monodispersity with high density of carboxyl groups,ultra-high fluorescent brightness and excellent fluorescent stability.QBs with the sizes ranged from 60-600 nm can be controllably prepared by varying the parameters of the ultrasonic power,the concentration of surfactant and the dosage of oil-phase solvent.The luminescent intensity of QBs was found to increase sharply with the increase of size because the larger QBs contained more QDs.2)The as-prepared QBs with the size of 255 nm were explored as novel fluorescent labels in the ICA with competitive and sandwich formats for sensitive detection of zearalenone?ZEN?and Plasmodium falciparum?pf?,respectively.The dynamic linear curves for ZEN and pf quantitative detection were obtained under optimal conditions.The proposed ICAs exhibited the high detection sensitivity for ZEN and pf quantitative detection,whose limit of detection?LOD?values were respectively 6-and 17-fold lower than conventional ICAs.In addition,these proposed ICAs for detecting real and spiked samples also showed excellent accuracy,repeatability and reliability.3)Improving the signal intensity of labeling probes is an important strategy to enhance the sensitivity of ICA.The larger-sized QBs possess higher fluorescent intensity because of their encapsulating more QDs into polymer matrix.However,the larger-sized QBs could decrease the immunoreaction efficiency of antigen-antibodies on NC membrane.To investigate the effect of the size of QBs on the sensitivity of ICA,a series of QBs with the size ranged from 60-600 nm were prepared and used as labels of a competitive ICA for OTA quantification and a sandwich ICA for HBsAg detection.It was demonstrated that the sensitivity of competitive ICA can be enhanced by using relatively larger QBs,whereas over-sized QBs would in turn decrease the sensitivity due to steric-hindrance effect.the medium-sized QBs?124 nm?were proved to be most suitable for establish competitive ICA with the best detection sensitivity.In addition,the pore size of NC membrane was considered as another important factor that influence the sensitivity of ICA because the pore size of NC membrane could affect the flow velocity of the probe on NC membrane,and thereby influencing the immunoreaction efficiency of the probes and antibodies on T line site.Thus,in sandwich ICA for HBs Ag determination,the effects of the size of labels and pore size of NC membrane on the sensitivity of sandwich ICA were further investigated.Results indicated that using 235nm QB as the probes,and the probes swimming on CN95 NC membrane?15?m pore size?,the sensitivity of sandwich ICA for HBsAg detection was 20-fold enhanced than that of conventional GNPs-ICA.In addition,compared with commercial GNPs-ICA,the proposed sandwich ICA also showed an excellent accuracy with 100%precision for detecting 90 real serum samples.4)Three QBs with distinguishable yellow,orange,and red luminescence were synthesized via encapsulating CdSe/ZnS QDs with 575 and 615 nm emissions with different ratio into polymer matrix,and the as-prepared tricolor QBs were used as fluorescent detection probes to develop a tricolor QB-based multiplex ICA?QB-m ICA?for the visual detection of OTA,FB₁,and ZEN.The visual detection limits of the developed QB-m ICA for OTA,FB₁,and ZEN under the developed conditions reached up to 5,20,and 10 ng/m L within 15 min,respectively.The accuracy,repeatability,selectivity,and practicability of our proposed QB-mICA were evaluated by using naturally contaminated maize samples.Also,a high correlation in simultaneously testing three mycotoxins was observed between the developed QB-mICA and the well-accepted ultra-performance liquid chromatography?UPLC-FLD?.In summary,some significant conclusions were listed as follows:1)The QBs with the size ranged from 60-600 nm was synthesized by a simple UESE method.The resultant QBs could be a promising label in ICAs due to its ultra-high fluorescent brightnes and good monodispersity.The LOD values for ZEN and pf determination in our developed QBs-ICA were 6-and 17-fold lower than that of the conventional GNPs-ICA.2)The effects of the fluorescent probe on the sensitivity of ICA with competitive and sandwich format were investigated for the first time,and the optimal size of QBs for competitive and sandwich ICA was 124 nm and 235 nm,respectively.This study provides an efficient supporting data for the rational adoption of the different size of fluorescent probe for competitive and sandwich ICA analysis.3)3)The tricolor QBs were obtained by a simple UESE method via adjusting the ratio of QD₅₇₅ and QD₆₁₅.The as-prepared QBs were used as labels to develop a multiplex QB-ICA method for the visual detection of OTA,FB₁,and ZEN in real maize samples.This proposed tricolor QB-ICA can serve as a promising and versatile multiplexed POCT tool for other targets analyzing.