Effects Of Different Sized Gold Nanoflowers On The Detection Performance Of Sandwich Immunochromatographic Assay

Immunochromatographic assay?ICA?is one of the most popular point-of-care testing products because of its unique advantages of rapidity?5¹5 min?,simplicity,low cost,and so on.Colloidal gold is a classical labeling material for ICA reporters on account of its facile synthesis and modification,good biological compatibility,and naked-eye detection.However,traditional colloidal gold based ICA that generally uses 20⁴0 nm spherical gold nanoparticles as signal reporters,has low sensitivity owing to its insuffcient brightness.In comparison to spherical gold nanoparticles,gold nanoflowers?AuNFs?with similar size have larger molar extinction coefficient and stronger signal intensity.Therefore,AuNFs as a novel signal reporter might improve the detection performance of ICA.Furthermore,the size of nanomaterials and porosity of the nitrocellulose?NC?membrane would affect the flowing of the labeled probe on the NC membrane,and further affect the binding efficiency of the probe and the antigen or antibody on the test line and the control line.Therefore,the purpose of this paper is to investigate the effects of the size of AuNFs and porosity of NC membrane on the detection performance of sandwich ICA.Firstly,the effects of AuNF size on the detection sensitivity of sandwich ICA were studied.AuNFs with five different sizes?33,47,79,152,and 195 nm?were synthesized,and then applied as reporters in ICA.Human Chorionic Gonadotropin?HCG?was selected as a model target analyte.When the optical intensities of five different sized AuNF probes are consistent,AuNFs with sizes of 47 and 79 nm exhibited the highest detection sensitivity with the limit of detection?LOD?for HCG at 9 mIU/mL.Futhermore,47 nm AuNFs were used as reporters for the preparation of HCG strip,and the detection performance of strip was evaluated.Two related calibration curves were presented for HCG quantitative detection.when the HCG concentration is in the range of 9 mIU/mL to 288 mIU/mL,the regression equation is y=0.4025 ln?x?-0.4483?R²=0.9872?,while the HCG concentration is from 288mIU/mL to 2304 mIU/mL,the quantitative analysis can be conducted according to the regression equation of y=1.0503 ln?x?-4.2393?R²=0.9831?.In addition,.the AuNF₄₇-ICA showed a weak cross reaction to its similar structures including luteinizing hormone and follicle stimulating hormone,and no cross reaction to other interfering proteins including carcinoembryonic antigen and bovine serum albumin.The intra-and inter-assay recoveries for HCG spiked serum samples were of 81.82%¹02.87%and 81.19%¹14.44%,respectively,and the variation coefficients were of 3.29%⁶.76%and 3.87%⁸.08%,indicating that the proposed method has an accepted accuracy.The analytical reliability of the AuNF₄₇-ICA was further confirmed with a commercial chemiluminesecence immunoassay?CLIA?kit,and the results revealed that the AuNF₄₇-ICA method is comparable with commercial CLIA kit for the quantitative detection of HCG.Secondly,79 nm AuNFs were used as the reporters of ICA for quantitative detection of Hepatitis B surface Antigen?HBsAg?,and the effects of the porosity of NC membrane on the detection sensitivity of ICA were discussed.Under the optimal conditions,using the CN170 membrane for running 79 nm AuNF probes,the strip showed the highest sensitivity for HBsAg detection with a LOD of 1 ng/mL,and the linear range for HBsAg quantitative detection was in the range of 1 to 5000 ng/mL.The corresponding regression equations were expressed as follows:y=0.0545 ln?x?+0.0023?R²=0.9520,1 ng/mL?x?50 ng/mL?and y=0.3418 ln?x?-1.1563?R²=0.9859,50 ng/mL?x?5000 ng/mL?.The LOD for HBsAg on CN140 and CN95were of 5 ng/mL and 10 ng/mL,respectively.The above results suggested that the NC membrane with the smaller pores is conductive to improving the sensitivity of the strip because the slower swimming speed of probes on NC membrane could increase the binding efficiencies of probes and antigens in the sample solution,as well as the probe-antigen complex and antibodies on the test line.The results of acceleration stability at 60?showed that the proposed AuNF₇₉-ICA could keep an excellent detection performance in a room temperature at least for two years.What's more,the proposed strips were used for the detection of real serum samples.Compared with the commercial strip products,the reuslts of AuNF-ICA method showed a higher?87.5%?coincidence with those of CLIA method because of its higher detection sensitivity.