Immunoassay Based On Pt/IrO₂ Nanoflowers For Detection Of Progesterone

Progesterone（P4）,a class of steroid hormones,plays an indispensable role in keeping human bodily and psychologically fitness.Because the concentration of P4 in human body is related to the incidence of diseases,it has a relatively important detection significance.At present,researchers have developed a variety of methods for the analysis of P4.Usage of non-invasiveness approaches,obtaining saliva,urine or milk samples for the determination of P4,have become one of the research hotspots.As one of the indicators,the concentration of P4 in human body can be applied to clinical diagnosis.Consequently,the establishment of a rapid,sensitive and high-throughput method for the detection of P4 has grabbed widespread attention.First,two progesterone derivatives,4-androsten-3-one-5-ene-17-carboxylic Acid and Progesterone 3-（O-carboxymethyl）oxime,were selected as haptens in this study.Taking advantage of carbon diimine method,immunogen（Progesterone-BSA）and coating antigen（Progesterone-3CMO-OVA）were prepared by coupling with bovine serum albumin（BSA）and ovalbumin（OVA）,respectively.Successful synthesis of Progesterone-BSA and Progesterone-3CMO-OVA were identified by UV-Vis absorption spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）.Immunological tests showed that Progesterone-3CMO-OVA was successfully prepared.Next,antiserum against P4 were obtained by immunizing New Zealand white rabbits with the prepared Progesterone-BSA,and then purified polyclonal antibodies were labeled with activated derivatives of biotin through chemical modification.The marker with catalytic function is a key factor affecting the sensitivity of detection.Finally,we incorporated Pt/IrO₂ nanocomposites（Pt/IrO₂ NPs）with copper（Ⅱ）ion,streptavidin（SA）and horseradish peroxidase（HRP）utilizing one-pot co-precipitation strategy to prepare a late-model hybrid nanoflowers that exhibited the dual functions of ability to specifically recognize biotin and activity on catalytic substrates.Transmission Electron Microscopy（TEM）,Scanning electron microscopy（SEM）,Dynamic Light Scattering（DLS）,X-ray photoelectron spectroscopy（XPS）,Fourier transform infrared（FTIR）and Energy-dispersive spectroscopy（EDS）were exploited in characterization.Thanks to specific antibody and Pt/IrO₂@SA@HRP nanoflowers,the developed ultrasensitive colorimetric assay showed a linear interval range from 0.217 ng/m L to 7.934ng/m L.The half maximal inhibitory concentration(IC₅₀)for monitoring P4 was 1.311 ng/m L and the limit of detection(LOD=IC₁₀)reached 0.076 ng/m L in the proposed assay,whose results were superior to traditional ELISA.Medial recoveries between 79.6 and 107%,and acceptable coefficients of variation below 10.6%could meet the requirements of the experiment.Saliva samples with diverse spiked levels using multiple-catalysis ELISA were verified by liquid chromatography-electrospray tandem mass spectrometry（LC-MS/MS）,which gave a good correlation.The effectiveness of the method was proved by the detection of real saliva samples.Collectively,results reveal that multiple-catalysis ELISA based on Pt/IrO₂ nanoflowers is expected to be a potentially effective tool for routine screening small molecule weight compounds in real samples.