| Polychlorinated biphenyls(PCBs) are a class of anthropogenic chlorinated organic compounds. Because of the desirable physical and chemical properties, PCBs have been widely used in industrial application. Although PCBs have been banned for about forty years, they are still routinely found in various environments. Because of the long term stability, bio-accumulation and high toxicity in the environment, PCBs can make great potential threat to human healthy and ecological environment. PCBs have been listed as one of the most important environmental monitoring objects in most of the countries.The determination of PCBs is mainly based on gas chromatographic methods. However, these methods are known to manifest underlying disadvantages, such as expensive, time-consuming, and complex sample pre-treatment demanding. Moreover, they are unsuitable for on-site rapid detection and high throughput screen. On the contrary, immunoassay are reliable, ease-of-operation, low cost and sensitive, especially, they can provide rapid and high throughput screen. Therefore, developing a rapid immunoassay for PCBs detection has important significance and broad application space.In this work, three polyclonal antibody, anti-PCB12 antibody, anti-PCB37 antibody, anti-PCB77 antibody, and one monoclonal antibody anti-PCB37 antibody were obtained. Based on the antibodies, ic-ELISA was developed for PCB12, PCB37, and PCB77 detection; biotinylated antibodies were prepared, and biotin-streptavidin amplified enzyme-linked immunosorbent assay(BA-ELISA) was developed for the three PCB congeners detection; gold nanoparticals(GNPs) were modified by antibodies and DNA to form different GNPs probes. The probes were used to replace the antibody-DNA conjugate. Based on these probes, an improved bio-barcode assay(BCA) based on rt-IPCR was constructed for PCBs detection. All of the methods were used to analysis real environmental samples, and good results were obtained. The methods developed in this work can be used to provide rapid, high throughput screening and reliable test results for PCBs analysis in environmental samples.The main conclusions of this work are summarized as follows:(1) Based on active ester method and mixed anhydride method, PCB12, PCB37 and PCB77 haptens were conjugated with BSA and OVA respectively to synthesize immunogens and coating antigens, and the conjugates were characterized by ultraviolet absorption spectra; three polyclonal antibodies, anti-PCB12 antibody, anti-PCB37 antibody and anti-PCB77 antibody were prepared by immune rabbit, all the antibodies have good specificity and high titer; A specific monoclonal antibody targeting PCB 37 was obtained through through immunization, cell fusion, hybridoma cell culture, collection of ascites, compared with the polyclonal antibody, it has much better specificity.(2) Based on the obtained antibodies, ic-ELISA was constructed for PCB12, PCB37 and PCB77 detection. The experimental conditions were optimised including the concentration of the coating antigen and antibody, the incubation time, the blocking buffer, the solvent, the pH of the assay buffer and the ionic strength. Under the optimised conditions, the LOD for PCB12, PCB37 and PCB77 detection based on polyclonal antibody were 0.016 μg L-1, 0.048 μg L-1, 0.063 μg L-1 respectively; based on the monoclonal antibody, the LOD for PCB37 detection was 0.027 μg L-1. The methods were used to analyze sediment samples, and all the recoveries were in the range of 81%-115%, coefficient of variation(CV) were below 15%.(3) BA-ELISA was developed based on biotinylated antibodies for PCB12, PCB37 and PCB77 detection. The experimental conditions were optimised including the concentration of the coating antigen and biotinylated antibody, the concentration of SA-HRP, the incubation time, the biotinylated antibody and SA-HRP reaction time, the solvent, the pH of the assay buffer and the ionic strength. Under the optimised conditions, the LOD for PCB12, PCB37 and PCB77 detection based on polyclonal antibody were 4.5 ng L-1, 17 ng L-1 and 18 ng L-1 respectively; based on the monoclonal antibody, the LOD for PCB37 detection was 8 ng L-1. The methods were used to analyze PCBs in hairtail samples, and all the recoveries were in the range of 81%-113%, coefficient of variation(CV) were below 15%.(4) Two GNPs probes, goat anti-rabbit IgG-GNPs-DNA and goat anti-mouse IgG-GNPs-DNA were prepared by coupling goat anti-rabbit IgG, goat anti-mouse IgG and DNA with 15 nm GNPs. The two probes were characterized by UV–vis optical spectra and TEM to make sure antibody and barcode DNA were labeled on the surface of GNPs.Based on the two probes, indirect compete bio-barcode assays based on rt-IPCR were developed for PCB12, PCB37, PCB77 and Aroclor 1248 determination. Coating antigens coated on PCR tubes were competed with analytes for the polyclonal antibody. GNPs probes were then fixed on the wall through the immune reaction of polyclonal antibody and goat anti-rabbit IgG. Barcode DNA(signal DNA) used as template DNA was released from the particle surface to PCR tubes through the initial heating procedure of real-time PCR. Afterward, real-time PCR was performed to quantify the barcode DNA directly in the tubes. And the targets analytes were detected indirectly by barcode DNA determination.The experimental conditions were optimised including annealing temperature, the primer concentration, the concentration of the coating antigen and antibody, the blocking buffer, the concentration of GNPs probes, and the reaction time of antibody and GNPs probes. By using the probe goat anti-rabbit IgG-GNPs-DNA and polyclonal antibodies, the LOD for PCB12, PCB37, PCB77 and Aroclor 1248 detection were 2.63 pg L-1, 4.35 pg L-1, 1.72 pg L-1, 10.20 pg L-1 respectively. By using the goat anti-mouse IgG-GNPs-DNA and anti-PCB37 monoclonal antibody, the LOD for PCB37 detection was 2.03 pg L-1. The methods were used to detect PCBs in larimichthys polyactis samples, and all the recoveries were in the range of 81%-112%, CV were below 15%.(5) Five antibodies were labeled on the surface of GNPs respectively with DNA to form GNPs probes, anti-PCB12 antibody-GNPs-DNA, anti-PCB37 polyclonal antibody-GNPs-DNA, anti-PCB37 monoclonal antibody-GNPs-DNA, anti-PCB77 antibody-GNPs-DNA and anti-Aroclor 1248 antibody-GNPs-DNA. The probes were characterized by UV–vis optical spectra and TEM to make sure antibody and barcode DNA were labeled on the surface of GNPs.Based on the five GNPs probes, direct compete bio-barcode assays based on rt-IPCR were developed for PCB12, PCB37, PCB77 and Aroclor 1248 detection. Coating antigen coated on PCR tubes were competed with analytes for the antibody assembled on the gold nanoparticles. Then the probes could be fixed on the wall through immune reaction of coating antigen and antibody. And other free probes were washed out by the washing stage. Barcode DNA were released from the probes and used as template DNA in the intial heating procedure of real time PCR. Then real time PCR was performed to quantify the barcode DNA directly in the tubes. And the targets analytes were detected indirectly by barcode DNA determination.The experimental conditions were optimised including the concentration of GNPs probes, the blocking buffer, the incubation time, the solvent, the pH of the assay buffer and the ionic strength. Under the optimised conditions, standard curves were constructed for different target analyte. The LOD of this method for PCB12, PCB77 and Aroclor 1248 detection were 4.36 pg L-1, 9.12 pg L-1, 2.55 pg L-1. The LOD for PCB37 detection were 4.64 pg L-1 and 3.15 pg L-1, which were obtained by using the polyclonal antibody and monoclonal antibody respectively. The methods were used to detect PCBs in hairtail samples, and all the recoveries were in the range of 81%-110%, CV were below 15%.In conclusion, ic-ELISA, BA-ELISA, indirect BCA based on rt-IPCR and direct BCA based on rt-IPCR were constructed respectively in this paper for PCBs determination in Environmental samples. The methods showed good stability, high sensitivity, rapid detection and ease-of-operation. Moreover, their results showed good correlation with GC-ECD. Therefore, they can used for PCBs detection with rapid and high throughout screening in real environmental samples and have good application prospect. |
PDF Full Text Request