Study On Construction Of A Bifunctional Osmium (Os) Nanozyme Based Folic Acid Immunosorbent Assay

Nobel metal nanomaterials have been a trendy research topic in the field of biochemistry,with a wide range of applications in numerous fields such as target detection,sterilisation,therapy and sensing.Among them,platinum group element nanomaterials have caught our attention for their outstanding performance in catalysis,drug delivery and treatment,photothermal sensing and conversion,and electrical signal sensing.Osmium(Os),a member of the platinum group elements,has been scarely studied in recent years.However,nanoparticles composed of Os that as single element or alloyed can exhibit superior enzyme-like activity,which has inspired great research interest to develop and utilize these enzyme-like activity of Os nanomaterials.With the advent of the national open birth policy,there is a burgeoning concern for the health of pregnant women and infants,where folic acid detection is an important indicator in the hospital.As one of the members from the vitamin B family,folic acid(FA)plays an important role in the synthesis of nucleic acids in the human body.Deficiency of folic acid can lead to neural tube abnormalities and megaloblastic anaemia,which can seriously affect the health of newborn babies and pregnant women.Excessive folic acid intake can also disrupt human body's absorption of vitamin B₁₂ and may even lead to cancer.At the same time,FA detection is not only limited to employ in regular tests in hospital but is also necessary in the food and drug industry,where it is extra added.Thus the development of a highly sensitive,accurate,immediate and rapid method for FA detection shows promising future.In this study,two methods were used to prepare complete antigens by attaching carrier proteins to the amino and carboxyl groups of FA,and the successful preparation of these two complete antigens(both immunogen and coating antigen)was identified using SDS-PAGE and MALDI-TOF.Secondly,the two immunogens were used to immunise New Zealand rabbits to obtain two polyclonal antibodies against FA(anti-FA-p Ab).The two coating antigen and the two antibodies were selected for subsequent immunoassay development,with the conclusion that each antibody was more sensitive for the analysis in the heterologous way.An osmium nanozyme(osmium nano-hydrangea,Os NHs)with an average particle size of 27 nm was prepared by a one-pot sythensis method.Fristly the Os NHs was characterized using TEM,EDS and XPS.Stability studies,mechanistic and kinetic studies on enzyme-like activity of Os NHs were also carried out and it turned out be an ideal peroxidase minic with great chemical and temperature stability,high catalytic efficiency and long-term storage stability,making it an impeccable alternative to HRP enzymes.Based on the excellent peroxidase-like activity performance of Os NHs and the selected combination of anti-FA-p Ab and coating antigen,an Os NHs@p Ab probe was prepared to establish a bifunctional immunoassay for FA detection.The enzyme-like activity of the Os NHs@p Ab was utilized to replace HRP in the traditional ELISA assay.An accurate and sensitive method for the quantitative assaay of FA(Os NHs-NLISA)was developed by catalyzing TMB to trigger a chromogenic reaction in ELISA,which shown a linear range of9.42-167.53 ng/m L,IC₅₀=39.73 ng/m L and the LOD=4.03 ng/m L in FA detection.An immunochromatographic test strip(Os NHs-LFIA)was prepared using a grey-black colour marker on Os NHs@p Ab to estabilish a sensitive,rapid and immediate qualitative FA assay,showing a visual threshold of 100 ng/m L and a visual detection limit of 0.01 ng/m L.Both methods showed high specificity for the FA detection and were also able to recognize FA in milk powder spiked samples.The average recoveries for the spiked samples ranged from93.9 to 103.6%with a coefficient of variation of less than 4.9%.These results were methodologically validated by UPLC-MS/MS and showed high correlation(R~2=0.999).In summary,the development of a bifunctional immunoassay model based on Os NHs can be used in a more sensitively,accurately and efficiently for FA detection method and is expected to be explored to become a novel assay for immediate detection techniques.