Development Of Novel Immunoassays Using Fluorescence Quenching And Dynamic Light Scattering As Signal Transducer

In recent years,public health issues,such as food safety,cancer diagnosis and disease prevention and control,have attracted much attention.The target objects associated with the health problems,such as mycotoxin,foodborne pathogen,cancer biomarker is hard to be perceived and identified as their small size and invisible morphology.Therefore,the establishment of quantitative and sensitive point-of-care testing methods for such target screening detection is an important and effective method to ensure public health.It is well known that the horseradish peroxidase-based enzyme-linked immunosorbent assay（HRP-ELISA）has been widely applied in the fields of food safety and environmental monitoring,disease diagnosis because of its advantages of easy operation,low cost and high throughput.However,the relatively low sensitivity of HRP-ELISA limits its application for the detection of target with trace concentration.Thus,it is of great significance to develop the immunosensors by introducing some novel signal transducers to realize the efficient,rapid and highly sensitive detection of target substances.In this study,we focus on the development of some novel immunosensors by using fluorescence quenching and dynamic light scattering as signal transducer for rapid,and sensitive detection of mycotoxin,food-borne pathogen,and biomarker.（1）A fluorescence quenching-based enzyme-linked immunosorbent assay（Fq-dcELISA）was established by using H₂O₂-sensitive CdTe quantum dots as the signal molecule,catalase（CAT）as an alternative to HRP,and the zearalenone（ZEN）labeled CAT as the competing antigen for the ZEN detection in corn samples.Under the optimal conditions,the limit of detection of proposed Fq-dcELISA reached at 4.1pg/m L in corn sample,and dynamic linear range for ZEN quantitative detection was2.4 pg/m L to 1.25 ng/m L.The half maximal inhibitory concentration of the proposed method was 75 pg/m L,which was 17 times lower than that of the conventional HRP-based ELISA.In addition,the proposed method showed no cross reaction with other common mycotoxins,and the average recoveries for inter and intra-assay ranged from 84.1%to 112.3%with the coefficient of variation below 5%.Moreover,the results from the proposed method also showed a significant correlation with those from the LC-MS/MS method.（2）A DLS-enhanced direct competitive enzyme-linked immunosorbent assay（DLS-dcELISA）was developed for the ultrasensitive detection of AFB₁ in corn.The DLS-dcELISA was established by using gold nanoparticle（GNPs）based light scattering as signal output,GOx as the replacement of HRP as AFB₁（aflatoxin B₁）carrier to prepare the competing antigen.GOx catalyzed glucose to produce H₂O₂,which was further converted into hydroxyl radicals in the presence of HRP and tyrosine to induce GNPs aggregation.The developed DLS-dcELISA exhibited ultrahigh sensitivity for AFB₁detection with a detection limit at 0.12 pg/m L,which was 153-and 385-fold lower than those of plasmonic ELISA and HRP-based dcELISA,respectively.This method showed no cross reaction with other common mycotoxins,and the average recoveries for inter and intra-assay ranged from 90.6%to 107.0%with the coefficient of variation below 15%.Moreover,the results from the proposed method also showed a significant correlation with the findings from the UPLC-FLD method.（3）Multi-branched gold nanoflowers（GNFs）with higher light scattering were used as the signal probes to enhance the detection performance of DLS immunosensor,and thiolated amphiphilic polymer was used to improve the colloidal stability and functionalize the antibodies on the surface of GNFs via ligand exchange method.Combined with our previously reported two-step large-volume immunomagnetic separation（IMS）method,the designed GNF＿DLS immunosensor achieved ultrasensitive detection of E.coli O157:H7 in pasteurized milk with no-wash operation.Under the optimal conditions,the proposed GNF＿DLS immunosensor provided an excellent linear detection for E.coli O157:H7 within the range from 6×10⁰ to 6×10⁴ CFU/m L,with a limit of detection of 2.7 CFU/m L.This method showed no cross reaction with other foodborne pathogens,and the average recoveries for inter and intra-assay ranged from 96.7%to 105.0%with the coefficient of variation below 15%.In addition,the proposed method also showed a great potential for several cell detection in 1 m L of sample solution,which was confirmed by the plate count method.（4）A novel DLS immunosensor system was developed by using functionalized M13 phage as recognition component to regulate the hydrodynamic diameter of GNFs,in which the functionalized M13 phage was obtained by the fused expression of mimic epitopes or nanoantibodies with pⅢprotein.Furtherly,the functionalized M13 phage was assembled on the surface of GNF probes through the antigen and antibody interaction,and thereby resulting the changes of the hydrodynamic diameter of GNFs.Under the optimal conditions,the proposed DLS sensor exhibited a high sensitivity for OTA detection in corn sample with a detection limit at 1.69pg/m L,and the average recoveries of intra and inter-assays were ranged from 94%to102%.The proposed method showed no cross-reaction with other common mycotoxins,and also exhibited good correlation with the results from LC-MS/MS method.In addition,the developed DLS immunosensor system also showed an accepted sensitivity for AFP detection in human serum with the detection limit of0.28 ng/m L.In order to remove the matrix interference,the gold magnetic nanoparticles were used as the alternative of GNFs to enrich the AFP from the real serum sample,and the detection limit of the proposed method for AFP determination was achieved at 0.057 ng/m L.Additionally,the proposed method showed no cross-reaction with other non-specific proteins and had a good correlation with the results of commercial chemiluminescence kit.