| Polycyclic aromatic hydrocarbons(PAHs)are an ubiquitous class of hydrophobic organic compounds consisting of two or more fused benzene rings in linear,angular,or cluster arrangements.Natural and anthropogenic processes can cause the production of PAHs.Many PAHs have been found to be teratogenic,carcinogenic and mutagenic.The abundance and toxicity of PAHs pose a great threat to human health.Up to now,the detection methods are mainly dependent on instrumental analysis,but these techniques require labor-intensive sample pre-treatments,expensive equipment and the applied reagents to be environmentally unfriendly.For these reasons,it is urgent to develop several more effective detection methods.In this study,phenanthrene(Phe),a common PAHs pollutants in water was used as analyte.Five immunoassays based on the specific reaction between antibody and antigen were developed for rapidly detection of Phe.The main research results of this paper are as follows:1.Preparation of Phe-protein conjugatesBy changing the molar ratios of Phe to the carrier proteins,2-Phenanthrene butanoic acid was conjugated with BSA and ovalbumin OVA by active ester method for immunogens(M1-6)and coating antigens(N1-3),respectively.The conjugates were confirmed qualitatively and the molecular conjugate ratio was analyzed by ultraviolet spectrophotometry.The coupling ratios of Phe-BSA and Phe-OVA were 1.2(M1),25.8(M2),30.2(M3),4.6(M4),11.6(M5),20.2(M6),3.6(N1),16.9(N2)and 25.7(N3),respectively.Balb/C mice were immunized with these immunogens for production of antibody against Phe.The immunogen M2 with coupling ratio of around 26 induced highest affinity antibody.2.Preparation and identification of the monoclonal antibody(Mc Ab)against PheM2 was used as immunogen to immunized Balb/C mice by footpad immunization method.After cell fusion,hybridoma selection and cloning,one hybridoma which stably secreted Mc Ab against Phe was obtained and named 1G7.The isotype,titer and average affinity of the Mc Ab were Ig G1,1: 6.4 ×105 and 3.29×108 L/mol,respectively.3.Development of an indirect competitive ELISA(ic-ELISA)for detection of PheAn ic-ELISA method for detection of Phe was developed by using the Mc Ab.The linear range of the assay was from 3.17 ng/m L to 100.13 ng/m L and the limit of detection(LOD)was 0.85 ng/m L.The recoveries of PAHs were ranged from 90.6 %-106.8 %,when the water samples were spiked with Phe at 10,40 and 80 ng/m L,respectively.4.Magnetic nanoparticles-phenanthrene conjugates(MNPs-Phe)probe based competitive chemiluminescence enzyme immunoassay(MNPs-ic CLEIA)for PheA MNPs-Phe hybrid conjugates probe was synthesized and MNPs-ic CLEIA was developed for rapid and sensitive detection of Phe in water samples.The linear range of the assay was from 1.85 ng/m L to 71.51 ng/m L and the LOD was 0.85 ng/m L.The sensitivity of the developed MNPsic CLEIA was about 3 times higher than that of the ic-ELISA.To fulfill the procedure,more than 90 min was saved compared with the ic-ELISA.The recoveries of PAHs from three different water samples were 97.2 %-103.4 %,95.8 %-104.8 % and 96.9 %-103.5 % respectively,which had a good correlation(R2 = 0.9986)with those from ic-ELISA.5.Fluorescence-linked immunosorbent assay(FLISA)for detection of PheA FLISA was developed using rhodamine B isothiocyanate(RBITC)as the model fluorescent dye conjugate Mc Ab for detection of Phe.The linear range of the assay was from 2.10 ng/m L to 91.95 ng/m L and the LOD was 1.05 ng/m L,which was approximately 2-fold lower than that of the ic-ELISA.Compared with ic-ELISA,more than 70 min was saved because of only one immunoreaction step was needed to accomplish the assay.The average recoveries of Phe from domestic water,contaminated water and natural water were 100.7 %,100.8 % and 101.2 %,respectively.There were good correlation between the two methods(R2=0.9948).6.Gold nanoparticles(Au NPs)-based fluorescence-activatable probe for detection of PheA fluorescence-activatable probe for detection of Phe was developed based on fluorescence resonance energy transfer(FRET)between RBITC and Au NPs.Under the optimal conditions,the linear range of the assay was from 46.22 pg/m L to 1787.62 pg/m L and the LOD was 21.36 pg/m L,which was approximately 100-fold lower than that of the ic-ELISA.Only 60 min was needed to fulfill the procedure.The recoveries of PAHs from domestic water,contaminated water and natural water were 94.4 %-106.3 %,95.9 %-107.6 % and 95.9 %-105.8 %,respectively.7.MNPs and Au NPs based dual nanoprobes assay for detection of PheA dual nanoprobes assay for detection of Phe based on MNPs-Phe probe and HRP-Au NPsMc Ab probe was established.Under the optimal conditions,the linear range of the assay was from 0.14 ng/m L to 6.55 ng/m L and the LOD was 0.08 ng/m L,which was approximately 30-fold lower than that of the ic-ELISA.Only 26 min was needed to fulfill the procedure,which was 5-fold shoter than that of the ic-ELISA.The recoveries of PAHs from domestic water,contaminated water and natural water were 90.5 %-109.8 %,91.2 %-108.0 % and 93.0 %-107.8 %,respectively. |
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