Development Of Quantum Dot Beads Immunochromatographic Assay For Quantitative Detection Of HBsAg And 25(OH)VD

Immunochromtogaphic assay?ICA?is a popular tool for point-of-care test because of its rapidity,acceptable anti-interference ability,and user-friendliness.In recent years,fluorescent probe based strip methods have been widely used for strip quantitative detection due to it's a relatively high sensitivety.Comparied with traditional organic dye,quantum dots?QDs?are regarded as promising fluorescent probes due to their unique properties such as size-tunable emission,broad adsorption,narrow and symmetric photoluminescence spectra,strong luminescence,and excellent anti-photostableaching property.In this paper,a quantum dot doped nanobeads?QBs?was utilized as immunochromatographic assay?ICA?signal probe of sandwich/competitive strip for detection of HBsAg and 25?OH?VD,respectively.The ratio of FI_T/FI_C was used to offset the effects of the inherent heterogeneity of test strips and the matrix containing the samples,and was suggested for stip quantitative detection.The detail contents are described as follows:First,the CdSe/ZnS doped quantum dot-beads was used as a highly luminescent probe in the sandwich-ICA for quantitative detection of HBsAg in human serum.The average diameter of the obtained QBs was 255 nm,and the luminescence intensity of QBs was presumably 2895 times brighter than that of the corresponding QDs.The optimal parameters of the QB based strip were as follows:the saturated amounts of the antibodies on the surface of QBs were 200?g mg^-1,the volume of QBs-anti-HBsAg-mAbs(0.3 mg mL^-1)for each strip was 1?L,the anti-HBsAg polyclonal antibodies(1.0 mg mL^-1)and donkey anti-mouse polyclonal antibodies(1.0 mg mL^-1)were suggested to dispense onto the NC membrane as test and control lines,respectively.The curve of the immunological kinetics was performed by plotting the values of FI_T,FI_C and FI_T/FI_C.against immunoreactions time,the results showed that the ratio of FI_T/FI_C based strip quantitative method can effectively offset the effect of the interpretation time on the stability of HBsAg quantitative analysis.In order to better explain the quantitative relationship between the ratio of FI_T/FI_C and HBsAg concentration,two linear independent regression equations were expressed with y=0.3361x-0.0059?R²=0.9983?for low HBsAg concentrations from 75 pg mL^-11 to 4.8 ng mL^-1,and y=0.8404 x-2.9364?R²=0.9939?in high HBsAg concentrations in the range of 4.8 ng mL^-11 to 75 ng mL^-1,and the QB based ICA method also exhibited a low detection of limit?LOD?for HBsAg at 75 pg mL^-1.Besides,the average recoveries of the intra-assay for HBsAg in real serum sample varied from 90.8%to 94.56%,with a CV ranging from 4.23%to 6.38%.The results of the inter-assay ranged from 90.14%to 94.76%,with a CV ranging from 0.23%to6.17%.These results revealed that the precision of the QBs-ICA method for HBsAg quantification was acceptable.In addition,47 HBsAg-positive serum samples were quantified using both QBs-ICA and a commercially available human HBsAg CLIA kit.Results were performed using a linear regression analysis y=1.3922 x+42.962?R²=0.9209,n=47?between two methods,indicating the proposed QBs-ICA can be used for rapid and quantitative detection of HBsAg in human serum.Meanwhile,a competitive QBs-ICA was also developed for rapid quantitative detection of 25?OH?VD.In order to decrease the non-specific absorption of QBs,in the NC membrane,150?g of anti-25?OH?VD complexes,which was composed of133.3?g of BSA and 16.7?g of anti-25?OH?VD mAbs,were conjugated to 1 mg of QBs.The 0.1 mg mL^-11 of 25?OH?VD-BSA and 0.5 mg mL^-11 of donkey anti-mouse polyclonal antibodies were dispensed onto the NC membrane as test and control lines,respectively.In addition,the effects of salt concentration,ethanol content of the sample extract,and interpretation time on the sensitivity and reproducibility of the QBs-ICA sensor were also explored.Under the optimal conditions,the QBs-based ICA sensor exhibited dynamic linear detection of25?OH?VD in PBS from 5 ng mL^-11 to 100 ng mL^-11 and the strip quantitative curevs could be represented by the following regression equation:y=-0.205 Lnx+1.3005?R²=0.9901,n=3?,where y is the competitive inhibition rate and x is the 25?OH?VD concentration.The IC₅₀0 of the QBs-ICA sensor was achieved at 49.6 ng mL^-1.The specificity of the QBs-ICA was evaluated by running several structurally related analogues including?25?OH?VD₂?25?OH?VD₃?1,25?OH?₂VD₃?1,25?OH?₂VD₂??VD₂ and VD₃.The results showed that the developed method exhibited 100%?28%?10%and 22%cross-reaction to 25?OH?VD₂,25?OH?VD₃,1,25?OH?₂VD₃ and 1,25?OH?₂VD₂,respectively,and no obvious?<1%?cross-reaction to VD₂ and VD₃.The recovery rates of the intra-and inter-assays for spiked samples ranged from 80.48%to100.65%,and a coefficients of variation were all below 10%.The QBs-ICA sensor has an acceptable accuracy for 25?OH?VD quantitative analysis.