| Perfluorinated compounds (PFCs) are a group of chemicals widely used for many applications, such as surfactants, stain repellents, corrosion inhibitors, fire-fighting foams and photolithographic film. To date, PFCs are released into the environment resulted in the widespread dispersal and accumulation in environment, as well as in human and ecological food chains. Among these pollutants, perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are mostly investigated due to their persistence and accumulation in environment and their potential toxicity via PPARα. The current detection and quantification of these compounds mainly depends on high performance liquid chromatography-mass spectrometry (HPLC/MS), which is a powerful, highly sensitive tool that allows determination of environmental concentrations of these compounds. However, HPLC/MS can only provide an accurate measurement of each of the known PFCs in sample extracts; it is unfavorable for high-throughput screening of large quantities of samples and not reliable for predicting the toxicity of complex mixtures of PFCs. Hence, a rapid and simple analytical method is desired for the screening of these compounds.This investigation aims at developing a bioassay method (called gold nanoparticle-PPRE probes assay) to detect PFCs, based on the interaction of PFCs (agonists of PPARα), PPARα, and PPARα-responsive elements (PPRE) on microplate. PPARαcould be activated, and then bind with PPRE probes. The probes modified by gold nanoparticle could be immobilized on the microplates through the PFOS-PPARα/RXRα-PPRE complexes system, thus be quantified through measuring the optical density after silver enhancement.Part I Gold nanoparticle-PPRE probes assay for identification and quantification of PFOS and other PFCsObjection: A novel bioanalytical method—gold nanoparticle-PPRE probes assay enhanced by silver, was established to detect and quantify of PFOS and other PFCs. Method: Among PFCs, PFOS as the most persistent end product of many other PFCs, which is mostly investigated due to its persistence and accumulation in environment and its potential toxicity via PPARα, was used as the main research object. In the present work, a method for determination and screening of PFCs was developed, based on the ligand-receptor interaction. PFOS firstly initiated the formation of PFOS-PPARα/RXRαcomplexes, which were then transferred into a microplate and captured via monoclonal anti-PPARαantibody linkage. The PPRE modified gold nanoparticle probes were added into the microplate and captured by the PFOS-PPARα/RXRαcomplexes. Subsequently, a silver enhancement step was applied for signal amplification because of the polymerizing properties of gold nanoparticles to Ag particles. With the signal magnification by silver enhancement, the results could be recorded more easily (the sensitivity could be increased). Since PPRE could only be recognized by activated PPARα/RXRαcomplexes in the reaction system, the quantity of PPRE modified nanoparticle probes captured by the PPARα/RXRαcomplexes would be proportional to the quantity of the ligand. Then the absorbance was detected, and the optical density, which was consistent with the concentrations of ligand-receptor complexes, was recorded by a microplate reader. Therefore, the dose-response relationship curve about the PFOS concentration against the optical density was obtained. The experiment conditions were optimized, and the results were compared with the standard detection methods HPLC/MS. Results: (I) From the western blotting results of nuclear protein extraction solution, the PPARαprotein density increased with the concentration of nuclear protein extract product increase. Thus, the nuclear receptor PPARαwas expressed highly in hepatocytes. (II) The optical density produced by gold nanoparticle-based silver enhancement could be observed in a dose-dependent manner with PFOS concentration, and the optimal silver-deposition time is 3 min. (III) The absorbance was linearly related with the PFOS concentration from 100 pM to 1μM (y = 0.2997x - 0.1531, R2 = 0.9582), EC50 was about 10nM and the detection limit was 10 pM at the signal to noise ration of 3. (IV) Quantification data of PFOS samples with good correlation between the PPRE modified nanoparticle probes bioassay and the HPLC/MS method, and there were no significant difference between them (P > 0.05). (V) Other PFCs (PFOA) were also indirectly quantified by this bioassay. Conclusion: Gold nanoparticle-PPRE probes assay is a sensitivie, rapid, simple and cost-effective method to detect the PFCs, which can be performed in common labratories.Part II Gold nanoparticle-PPRE probes assay for detection of PFCs in environmental and biological samplesObjection: To detect the TEQs of PFCs in the environmental and biological samples using gold nanoparticle-PPRE probes assay. Method: The TEQs of PFCs in the environmental samples (water from Yangtze River, Han River and East Lake in Wuhan) and biological samples (serum of rat administrated by PFOS) were detected quantitatively. After the extraction, concentration and purification, the samples were dissolved in DMSO and analyzed by gold nanoparticle-PPRE probes assay. And a series of tests of quality control and quality assurance were also conducted, including recovery, precision, accuracy, detection limit, linear range. Results: For detection of standard of PFOS, the detection limit of the bioassay was 10 pM, which was much lower than that of HPLC/MS, with a wide linear range (from 100 pM to 1μM for PFOS) and good accuracy, precision and recovery, which achieve quality control and quality assurance requirements of the basic biological detection technology. The results were compared with those of HPLC/MS method for water spiked samples with a significant correlation (r = 0.9893). The TEQs of samples of water analyzed by gold nanoparticle-PPRE probes assay were slightly higher than those of HPLC/MS method, and the difference was within the range of 10 times, since there were few of other PFCs existed in the samples. Conclusion: The gold nanoparticle-PPRE probes assay is a novel and valid bioanalytical method to detect the PFCs in environmental and biological samples.In conclusion, this study provides a novel, valid, cell-free and high-throughput bioanalytical method for PFCs in microplate, based on silver enhancement of gold nanoparticles and the interaction among ligands, PPARα, and PPRE on microplate, with higher sensitivity and wider linear range compared with traditional method. There are several advantages of this biological method. Firstly, it is performed in 96-well microplate, so it could be used for screening many environmental samples simultaneously. Secondly, with a wider linear range, it is less laborious than HPLC/MS methods; and as a biological method for measuring and screening PFCs from samples, it will only cost several hours to finish the protocol if the preparation work is sufficient. Thirdly, the method is promising for on-site measurement. It can be used to prescreen and analyze PFCs in environmental and biological samples, and serve as an assistant of HPLC/MS for prescreening of PFCs. |
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