Preparation Of Emulsion Self-assembly Based Optical Probe And Its Application In Lateral Flow Immunoassay

Lateral flow immunoassay（LFIA）has been widely applied in food safety,clinical diagnosis,environmental monitoring because of its numerous merits,such as rapidity,simplicity,low cost,and user-friendliness.However,the conventional colloidal gold based LFIA（AuNP-LFIA）often suffers from relatively low sensitivity,and inaccurate quantification,and cannot meet the requirements of precise quantitative detection in various fields.The usage of the probes with higher optical intensity instead of traditional AuNPs is one of the important strategies to improve the detection sensitivity of LFIA.Therefore,in this study,a serial of optical probes with high optical performances were designed and constructed,and their applications in LFIA were also proposed and investigated.（1）Preparation of high-performance colorimetric probe and its application in LFIA.AuNPs with the size of 20-40 nm is one of the most widely used colorimetric probes.However,the AuNPs based LFIA commonly suffers from low sensitivity because of the low molar extinction coefficient of AuNPs with size of 20-40 nm.Large-sized AuNPs has higher molar extinction intensity,which can effectively improve the detection sensitivity of LFIA,whereas the over-sized AuNPs（≥180 nm）are not conducive to enhancing the sensitivity of LFIA.In the chapters two and three of this thesis,we focused on the design of high-performance colorimetric probes and their application in LFIA.Firstly,the contribution of molar extinction intensity(Q_ext),light absorption cross-section(Q_abs)and light scattering cross-section(Q_sca)of AuNPs on the signal output of LFIA were investigated.Secondly,the colloidal gold microspheres（AuSAs）with strong absorption and low scattering were prepared by encapsulating a numerous small sized AuNPs（10 nm）in polymer by microemulsion self-assembly method.On this basis,the photophysical properties of AuSAs and AuNPs with different sizes were compared,and the effects of AuSA size on the detection sensitivity of LFIA were further investigated.The results show that:1)the light absorption rather than light scattering of probes dominated the signal readout of colorimetricLFIA;2)The Q_ext of AuSAs increased with the size increasing in a cubic relationship,and the OD_abs of AuSAs with the size large than 180 nm is remarkably higher than that of AuNPs with the same size.3)In the sandwich LFIA,AuSAs with size of 270 nm were used as probes,and the sensitivity of the LFIA for detection of Human Chorionic Gonadotropin（HCG）reached at 0.45 m IU/m L,which is 40 times higher than that of the traditional AuNP₄₀-LFIA.But,the over-sized AuSAs（400 nm）would produce serious nonspecific binding on NC membrane,which could reduce the detection sensitivity of LFIA.4)In the competitive LFIA,the AuSAs with size of 129nm were used as probes to realize the highly sensitive detection of FB₁.Compared with the traditional AuNP₄₀-LFIA,the naked-eye qualitative and instrumental quantitative sensitivities of AuSA-LFIA were improved by 8-and 4-times,respectively.（2）Preparation of highly luminescent aggregation-induced emission self-assembling microspheres（AIESAs）and their applications in LFIA.Compared with colorimetricLFIA,fluorescent LFIA has better sensitivity and anti-matrix interference ability.However,the traditional organic dyes were commonly troubled by the defect of aggregated fluorescence quenching（ACQ）in the preparation of fluorescent microspheres.In theory,aggregation-induced luminescent dyes（AIEgens）are ideal fluorescence substrates for developing high-performance fluorescent microspheres.Therefore,in the chapter four,green-emissive AIE fluorescent microspheres（GAIESAs）were prepared by emulsion self-assembly method,and as-prepared GAIESAs has been used as signal probes to construct competitive LFIA（GAIESA-cLFIA）for the sensitive detection of Ochratoxin A（OTA）.Under the optimum conditions,the proposed GAIESA-cLFIA shows a high sensitivity for OTA quantitative determination with a dynamic linearity from 0.073 ng/m L to 1.61 ng/m L,and the standard curve can be expressed by the following equation:y=-0.194ln（x）+0.2927（R²=0.9935）with the IC₅₀ and LOD values at 0.344 ng/m L and 0.043ng/m L,respectively.The average recoveries of OTA spiked corn sample ranged from82.6%to 116.5%with the CV in a range of 7.83%to 14.77%,indicating that the proposed GAIESA-cLFIA has excellent accuracy for OTA quantitative determination in real corn samples.The accelerated aging experiment at 60℃indicated that the shelf life of the developed GAIESA-cLFIAs was more than one year.In the chapter five,four kinds of red-emitting AIESAs（RAIESAs）with sizes of150,250,350 and 450 nm were prepared by emulsion self-assembly method.And the effects of the RAIESA size on the sensitivity of sandwich LFIA（sLFIA）were investigated.The results showed that the fluorescent signal intensities of RAIESAs increased with the RAIESA size increasing,and the fluorescent intensities were proportional to the third power of their bulk.Secondly,the sensitivity of the RRAIESA-sLFIA for the detection of serum inflammatory factor procalcitonin（PCT）increased with the increase of the RAIESA size.However,oversized RAIESAs（such as 450 nm）are prone to settle on the NC membrane,which can cause a strong nonspecific absorption of the RAIESA probes on the T line.The best sensitivity（LOD=6.33 pg/m L）was achieved by using 350 nm RAIESAs as the signal probes,which were 10.6-,2.29-and 4.82-times higher than those of 150,250,and 450 nm RAIESAs,respectively.In chapter six,a variety of AIESAs with the fluorescence emission wavelength ranging from 480 nm to 600 nm were prepared via adding different polymers or adjusting the ratio of polymer and AIEgen during the emulsion self-assembly process.Subsequently,the AIESAs with five fluorescent emissions of red,orange,yellow,green,and cyan were selected as signal probes to develop a multicolor AIESAs-based multiplex LFIA（m AIESA-m LFIA）for the visual and quantitative detection of AFB₁,OTA,ZEN and FB₁.The cut-off values of the proposed LFIAs for the AFB₁,OTA,ZEN and FB₁determination by naked eye were 1,4,5 and 2 ng/m L,respectively.The q LODs obtained by the smartphone software for the AFB₁,OTA,ZEN and FB₁quantitative detection were 0.007,0.024,0.07,and 0.079 ng/m L respectively.In addition,the multicolor AIESAs also exhibits the broad application prospect in the field of color writing and printing.（3）Preparation of colorimetric-fluorescent multifunctional nanocomposites（PFNPs）and their application in LFIA.In this chapter,the colorimetric-fluorescent multifunctional nanocomposites with different structures including“core-shell-shell”,“core-shell”,and“Janus”structures were prepared by co-assembling the 10 nm AuNPs and AIEgens into PAMO polymers,which was obtained by regulating the aggregation state and spatial distribution of AuNPs in the nanocomposites.On this basis,the effects of the geometric distribution of AuNPs in the nanocomposites on their plasma activity and fluorescent signal intensity were systematically investigated and discussed.The dissipative particle dynamics model was used to elaborate the inherent law of the ligands on the surface of AuNPs driving phase separation for regulating the geometric structure of AuNPs in the nanocomposites.Finally,the as-prepared PFNPs were used as the probes of LFIA for the simultaneous detection of PCT and CRP.The cut-off limits of the developed LFIA by naked-eye detection were0.048 ng/m L and 3.9 ng/m L,respectively,and the LODs for the quantitative detection of PCT and CRP by the strip reader were 0.0074 ng/m L and 0.37 ng/m L,respectively.In addition,the as-prepared PFNPs with the“Janus”structure was also used to establish a LFIA for simultaneously detecting and instantaneously killing Escherichia coli O157:H7 by utilizing the plasma-fluorescence-photothermal activities of PFNPs.To sum up,in this paper,the AuSAs with high light absorption but low light scattering was prepared by the microemulsion self-assembly method.The as-prepared AuSAs overcomes the shortcoming of large-sized AuNPs with high light scattering because it favors the absorption-dominated signal output in LFIA,and this study provides a promising strategy for constructing high-performance colorimetric probes.Subsequently,a series of novel fluorescent probes and colorimetric-fluorescent probes based on aggregation-induced luminescent materials were prepared,and this study can provide the basic guidelines for the design and construction of high-performance fluorescent probes and their advanced applications in LFIA.