Research On Detection Methods For Acenaphthene And Fluorene Based On Fluorescent Microspheres And Gold Nanorods

Polycyclic aromatic hydrocarbons?PAHs?comprise a class of organic compounds composed of two or more fused aromatic rings.The United States Environmental Protection Agency?USEPA?has identified 16 PAHs as priority pollutants because of their carcinogenic and mutagenic properties.At present,PAHs are analyzed by instrument.These instrumental techniques require complicated sample preparation steps,sophisticated equipment and skilled personnel.Therefor,there is an emergent need for simple,and sensitive detection methods for PAHs.Recently,nanomaterials have been widely engaged in analytical science,including micro nucleic acid and small molecular?micRNA,RNA,pesticide,mycotoxins and antibiotic?and protein?tumor markers,viral protien and bacterial proteins?,which promotes the innovation and development of the detection technology.In this paper,we developed simple,rapid and sensitive detection methods based on antigen-antibody reaction and nanomaterials.The main contents and conclusion of this paper are as follows:1.Preparationofcoatingantigen?ALA-OVA,ALArepresent5-Acenaphthenecarboxylic acid,which was analogue of acenaphthene?and immunogen?ALA-BSA?of acenaphthene by mixed anhydride method and coating antigen?FGL-OVA,FGL represent Fmoc-Gly-OH,which was analogues of fluorene?and immunogen?FGL-BSA?of fluorene by active ester method.Then,Immunogen was emulsified by IKA emulsified device.After cell fusion polyethylene glycol?PEG?and screening by limiting dilution analysis?LDA?with HAT medium,a monoclonal cell line?D8?,excreting anti-acenaphthene antibody and three monoclonal cell line?B7,E7and E10?,excreting anti-fluorene antibody were obtained.D8 exhibited affinity for acenaphthene of 5×10¹⁰ L/mol,the subtype of D8 was IgG2b.E7exhibited affinity for fluorene of1.8×10¹⁰ L/mol.The subtype of B7,E7and E10 were IgG1.2.Based on the monoclonal antibody?D8?,an indirect competitive enzyme-linked immunosorbent assay?Ic-ELISA?was developed for acenaphthene in water.Several physicochemical factors?concentrations of coating antigen and monoclonal antibody,blocking solution,reaction time of primary antiobdy,second antibdy and substrate?that influence assay performance were optimized.The linear range of the assay was between 3.53 ng/mL and 41.98 ng/mL.The limit of detection?LOD?was2.33 ng/mL.The recovery ranged from 91.52 to 117.28%in the water.D8 exhibited cross-reactivity to anthracene,phenanthrene,pyrene,fluorene,fluoranthene and benzo[a]pyrene and no corss reactivity with other 9 PAHs.3.Based on the monoclonal antibody?E7?,an indirect competitive enzyme-linked immunosorbent assay?Ic-ELISA?was developed for fluorene in water.Several physicochemical factors?concentrations of coating antigen and monoclonal antibody,blocking solution and reaction time of primary antiobdy,second antibdy and substrate?that influence assay performance were optimized.The linear range of the assay was between 0.96 ng/mL and 302.29 ng/mL.The limit of detection?LOD?was 0.37ng/mL.The recovery ranged from 81.88%to 104.96%in the water.E7 exhibited no cross-reactivity with other 15 PAHs.4.The genes encoding light chain and heavy chain of anti-fluorene monoclonal antibody were cloned by RT-PCR based on the monoclonal antibody cell line of B7,E7 and E10.There remains small difference in bases and amino acids of light chain and obvious difference in bases and amino acids of heavy chain,which demonstrate the higher sensitivity of E7 than B7 and E10.Finanlly,we acquired anti-fluorene?E7?single chain variable fragment?ScFv?gene.The heavy chain and light chain of ScFv to fluorene hold three complementarity-determining region?CDR?and four framework region?FR?in the McAb from murine,respectively.The homologous rate was 92.98%between the gene of heavy chain and IMGH1-80*01?AC160990?.The homologous rate was 94.74%between the gene of light chain and IGKJ2*01.It is known that the molecular weight is 25800.63,the molecular formaula is C1134H1736N306O366S9,the isoelectric point is 8.20,the hydrophilic index is-0.476,the fat index is 60.13.5.Preparation of fluorescent probes by mixed anhydride method based on the monoclonal antibody?E7?and Eu-COOH fluorescent microsphere.A sensitive time-resolved fluorescence immunochromatographic assay for fluorene was developed for the first time.The linear range of the assay was between 0.98 and 62.50ng/mL.The limit of detection?LOD?was 0.09 ng/mL.The recovery ranged from84.50%to 102.90%in the water.6.A sensitive time-resolved fluorescence Ic-ELISA for fluorene was developed for the first time based on the preparation of fluorescent probes.The linear range of the assay was between 0.032 and 0.587 ng/mL.The limit of detection?LOD?was0.019 ng/mL.The recovery ranged from 81.00%to 106.00%in the water.7.A plasmonic Ic-ELISA for fluorene was developed based on the monoclonal antibody?E7?and gold nanorods?longitudinal absorbance bands at 670 nm?using seed-mediated growth method.Firstly,the ALP bound on the polystyrene microwells based on antigen-antibody reaction and will hydrolyze ascorbic acid 2-phosphate into ascorbic acid due to the substrate dephosphorylation.secondly,KIO3 is reduced into to iodine due to AA and then etches gold nanorods from rod to sphere in shape.However,the etching was inhibited by adding the McAb against fluorene and fluorene.The reason was fluorene in samples competed with FGL-OVA bound on the polystyrene microwells to bind with McAb,which lead to the reduction of ALP bound on the polystyrene microwells and iodine.With the increase of concentration of fluorene,the shape change of gold nanorods contribute to a red-shift of longitudinal localized surface plasmon resonance?LSPR?with a significant color change from pink to blue.The linear range of the assay was between 0.030 and 2.00 ng/mL.The limit of detection?LOD?was 0.024 ng/mL.The recovery ranged from 73.00 to88.50%in the water.8.A new plasmonic Ic-ELISA for fluorene was developed based on deposition of silver on gold nanorods due to catalysis of enzyme and the monoclonal antibody?E7?and gold nanorods?longitudinal absorbance bands at 710 nm?.Firstly,the ALP bound on the polystyrene microwells based on antigen-antibody reaction.Subsequently,ALP hydrolyze p-aminophenyl phosphate?pAPP?into 4-aminophenol due to the substrate dephosphorylation.However,the etching was inhibited by adding the McAb against fluorene and fluorene.The reason was fluorene in samples competed with FGL-OVA bound on the polystyrene microwells to bind with McAb,which lead to the reduction of ALP bound on the polystyrene microwells and 4-aminophenol.With the increase of concentration of fluorene,the shape change of gold nanorods contribute to a red-shift of longitudinal localized surface plasmon resonance?LSPR?with a significant color change from green to pink.The linear range of the assay was between 0.063 and 4.00ng/mL.The limit of detection?LOD?was 0.035 ng/mL.The recovery ranged from83.00%to 96.00%in the water.In a word,five methods of immunoassay for fluorene were established in this paper.The detection time of fluorene based on iodine-etched GNRs-P-ELISA,based on silver deposition GNRs-P-ELISA,traditional Ic-ELISA,time-resolved fluorescence immunoassay and time-resolved fluorescence immunochromatography were 6 hours,6 hours,5 hours,4 hours and 0.5 hours,respectively.Compared with traditional Ic-ELISA,the sensitivity of other four 4 immunological methods for fluorene meets the USEPA standard?0.2 ng/mL?.The detection time of developed time-resolved fluorescence immunochromatography only taked 0.5 hours,which provide a rapid detection method for fluorene.