Fluorescence Polarization Assay Based On PPARα For The Detection Of Phthalate Esters

Phthalate esters(PAEs) have been widely used as plasticizers. As the PAEs are not chemically bound to plastic, they can migrate into the environment, cause pollution, and be hazardous to health. PAEs are mainly detected by chromatographies and immunoassays at present. The former can only be conducted in the laboratory and the latter focus on detecting one PAE or several PAEs, both of which have limitations. Nowadays, there is an urgent need to develop a broad-spectrum, sensitive, fast, and on-site method for monitoring multiple PAEs simultaneously to reduce the time and the cost of detection, which has important significance for assurance of food safety.In this work, a fluorescence polarization assay based on peroxisome proliferator-activated receptors(PPARs) has been developed for the determination of PAEs in Chinese spirits. The receptors could recognize a class of objects. Moreover, it’s easier to prepare large scale receptors than antibodies. Therefore, it is suitable for monitoring multiple PAEs simultaneously by using receptors, which has more practical significance for evaluation of total toxic equivalent of PAEs mixture.Based on the high affinity for PAEs, mPPARα was investigated in this work. In order to improve the efficiency of soluble expression, the protein was modified with computer-aided molecular modeling design. The deletion of N-terminal amino acids from 202 to 266 was conducted to produce a new soluble protein named mPPARα-LBD*. The mPPARα-LBD* cDNA fragment was amplified from cDNA generated from mouse liver mRNA by RT-PCR, and then cloned into pET28 a vector. The pET28a-mPPARα-LBD* plasmids were transformed into Escherichia coli strain Rosetta(DE3). Expression of the mPPARα-LBD* protein was induced with IPTG. The supernatant was applied to a Ni-NTA column, then washed and eluted with buffers. A high level of expression of an induced protein was achieved. Western blot analysis confirmed the predominant protein band corresponded to His-tagged mPPARα-LBD*. Concentration of the recombinant protein after concentration was 0.33 mg m L-1. The ability of the recombinant protein binding ligand was verified with saturation experiment of the probe and mPPARα-LBD*.The fluorescence polarization assay was based on mPPARα-LBD* and C4-BODIPY-C9. Optimal reaction conditions for FP assay were 10 min of incubation with a probe concentration of 40 nM and a protein concentration of 80 n M. FP assay showed that PAEs compete for the binding sites in a concentration-dependent manner. The limits of detection for 10 PAEs were 0.7-11.2 μM, where the limits of detection for DEHP and DBP were lower than the safe concentration, respectively. In order to avoid interference of media, the ethanol in Chinese spirits was removed by heating in water bath. The average recoveries of DEHP and DBP spiked in Chinese spirits were 80%. The coefficient variation was shown below 10%. FP assay has good repeatability, thus it is fit for detecting PAEs in Chinese spirits.Molecular docking approach was performed to explore the probable binding mode between PAEs and mPPARα-LBD* for better understanding their interactions. The structure of mPPARα-LBD was constructed by homology modeling using hPPARα-LBD as a templet. The binding of PAEs to mPPARα-LBD* occurs mostly through hydrogen bond and hydrophobic interaction. The π-π stacking interactions between benzene rings of PAEs and aromatic rings of F82, Y123, and Y273 were observed. Meanwhile, the hydrophobic interactions between benzene rings and F82 were also observed. H249 can form hydrogen bonds with the carbonyl oxygen of both ester groups of PAEs. Interestingly, S89 makes a hydrogen bond interaction with the alkoxyl oxygen of one ester group of DBP, while DEHP do not. The hydrophobic interactions between the hydrophobic channel and the alkyl chains of PAEs were observed to stabilize the PAE-mPPARα-LBD* binding. The binding abilities of long chain alkanes, branched alkanes, and benzene ring are stronger than the binding abilities of short chain alkanes, straight alkanes, and cyclohexane, respectively. The superposition effect of the hydrophobic interactions between the channel and the alkyl chains of PAEs contributes more to the receptor-ligand binding than hydrogen bond.On the basis of the research of molecular docking, the binding free energy between PAEs and mPPARα-LBD* was calculated. The molecular docking scores are in good agreement with the Kd values of PAEs(R2=0.948). Quantitative structure activity relationships model was developed. Based on the binding sites and docking scores, the binding abilities of novel ligands to PPARs could be predicted rapidly in theory. It would help to assisted screening new, biologically inert plasticizers, and has important significance for improving the screening efficiency and reducing work quantity.To summarize, a receptor-based fluorescence polarization assay for the determination of PAEs in Chinese spirits has been developed, which might be extended to detect other PPARs reactive pollutants in various matrices. FP assay are processed in a homogeneous system, dispensing with extra elution steps. After water bath heating treatment, Chinese spirits could be determined within 10 minutes. The method, which can be used for on-site determination by a portable fluorescence microplate reader, is suitable for high throughput screening in basic units.