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Oxidative stress is a unifying mechanism relating to the adverse effects induced by atmospheric particulate matter (PM). Owing to complex compositions, the role of PM constituents in triggering oxidative stress remains unclear. We employed synthetic particle suspensions with controlled sizes and compositions as PM surrogates to identify the key contributors to oxidative stress. The suspensions consisted of carbon black (CB) particles, polycyclic aromatic hydrocarbon adsorbed on CB, and dissolved metal ions. After the exposure of A549 or BEAS-2B cells to CB particles, the reactive oxygen species (ROS) level increased in a manner that was dependent on the size and concentration of the particles, in the order of 14 nm > 56 nm > 260 nm. Benzo[a]pyrene-coated CB also increased the ROS level, but not as much as the naked CB, and the benzo[a]pyrene-dione coating had no effect. The co-exposure of CB with one of the eight metal ions did not lead to any additional ROS increase. The results indicate that among the three major constituents, CB particles, and the small-sized ones in particular, contributed most to ROS generation. In mechanistic studies, damaged mitochondria cristae were observed, suggesting that mitochondria are one of the target organelles. Both naked and BaP-coated CB decreased the mitochondrial membrane potential and changed the mRNA expression of fusion/fission proteins. ROS inhibition experiments suggest that complex I of the mitochondria was involved, but complex III and the NADPH oxidase (NOX) system were not. It was thus postulated that small-sized particles could enter cells and disrupt the mitochondria, particularly complex I, leading to the elevated generation of ROS. Our work calls for more attention to be paid to the toxicity of the ultrafine fractions of PM.

Despite many studies on the toxicity of nanoplastic particles (NPPs) to aquatic invertebrates, the effects of ecological constituents such as humic substances (HSs) are often neglected. In our study, Daphnia magna was used to evaluate the effects of three HSs, natural organic matter (NOM), fulvic acid (FA), and humic acid (HA), on NPP toxicity and corona formation. Acute toxicities of NPPs were reduced by all HSs at environmentally relevant concentrations. NPPs elicited the upregulation of all genes related to detoxification, oxidative stress, and endocrine activity after 7 days of exposure. The presence of NOM or HA resulted in the mitigation of gene expression, whereas significantly higher upregulation of all of the genes was observed with FA. The presence of FA led to increased protein adsorption on NPPs in D. magna culture medium (eco-corona, EC) and homogenates (protein corona, PC), while there was less adsorption in the presence of HA. The highly abundant proteins identified in EC are involved in immune defense, cell maintenance, and antipredator response, while those in PC are responsible for lipid transport, antioxidant effects, and estrogen mediation. Our findings revealed the key influence of HSs on the toxicity of NPPs and provide an analytical and conceptual foundation for future study.

The growing evidence of the microbiome's crucial role in human health and disease has prompted research on understanding the impacts of engineered nanomaterials (ENMs) on commensal microorganisms. Accordingly, the number of studies addressing the ENM effects on intestinal microbiota has been rapidly increasing over the past few years. The focus on the gut microbiota is justified due to established metabolic and immunological functions of gut microbes, however, respiratory tract and skin microbiota also play important roles in host health and immunity. Here we review the composition and functions of microbiota inhabiting the major human organs that are prone to ENM exposure – skin, respiratory and digestive tract. We discuss the mechanisms of ENM actions relevant to physiological conditions and microbiota, and describe recently developed in vitro models for elucidating the ENM impacts on the gut microbiota. We find that studies pertinent to the oxygen levels prevailing in the digestive tract, are still limited. Finally, we analyze the results of in vivo studies conducted in vertebrate models – zebrafish, mice and rats – exposed to ENMs via ingestion. The relative abundances of bacterial phyla indicate that ENMs have a potential to modulate intestinal microbiota and induce harmful or beneficial effects in the host. Future perspectives include identification of the factors driving ENM-caused dysbiosis and the ENM impacts on microbiota with deviated composition, e.g., under compromised health conditions. Understanding the interplay between ENMs and the human microbiota is crucial for the continued development of nanomedicine and protection of human health against accidental exposure to hazardous ENMs.

As alternatives to perfluorooctanoic acid (PFOA), hexafluoropropylene oxide (HFPO) homologues, including hexafluoropropylene oxide dimer acid (HFPO-DA), hexafluoropropylene oxide trimer acid (HFPO-TA), and hexafluoropropylene oxide tetramer acid (HFPO-TeA), have been used in the fluoropolymer industry for a long period of time. These compounds have attracted widespread attention in recent years due to their environmental ubiquity and high bioaccumulation capability, as well as their toxicity. In our study, we evaluated the potential estrogenic effects of HFPOs in comparison to PFOA by ligand binding, transcriptional activity, and in vivo assays. Fluorescence ligand binding assays showed that both HFPO-TA and HFPO-TeA exhibited higher binding affinity to estrogen receptor ligand binding domains (ER-LBDs) than PFOA, with 2.5- and 57.5-fold higher affinity to ERα-LBD and 2.6- and 41.8-fold higher affinity to ERβ-LBD, respectively, whereas HFPO-DA exhibited weaker binding affinity than PFOA. Unlike PFOA, HFPO-TA and HFPO-TeA exhibited antagonistic activity toward the ERs' signaling pathway, with HFPO-TeA displaying the strongest potency. In silico study revealed that while PFOA binds with ERs in a similar fashion as 17β-estradiol, the HFPOs display an antagonistic binding mode. Using a zebrafish model, we further found that exposure to HFPO homologues significantly altered the levels of sex steroid hormones and vitellogenin. In general, both in vivo and in vitro results indicate that HFPO homologues might exert higher estrogenic effects than PFOA.

Many environmental chemicals have been found to exert estrogenic effects in cells and experimental animals by activating nuclear receptors such as estrogen receptors and estrogen-related receptors. These compounds include bisphenols, pesticides, polybrominated diphenyl ethers (PBDEs), organophosphate flame retardants, phthalates and metalloestrogens. G protein-coupled estrogen receptor (GPER) exists widely in numerous cells/tissues of human and other vertebrates. A number of studies have demonstrated that GPER plays a vital role in mediating the estrogenic effects of environmental pollutants. Even at very low concentrations, these chemicals may activate GPER pathways, thus affect many aspects of cellular functions including proliferation, metastasis and apoptosis, resulting in cancer progression, cardiovascular disorders, and reproductive dysfunction. This review summarized the environmental occurrence and human exposure levels of these pollutants, and integrated current experimental evidence toward revealing the underlying mechanisms of pollutant-induced cellular dysfunction via GPER. The GPER mediated rapid non-genomic actions play an important role in the process leading to the adverse effects observed in experimental animals and even in human beings.

With worldwide environmental accumulation of plastics and their recognized degradation into smaller particles, attention to their impacts on ecological systems and humans has been increasing recently. However, environmental factors and their impacts are seldom considered during their eco-toxicity evaluation. In this study, D. magna neonates were used to assess and compare the acute toxicities of polystyrene microplastic particles (MPPs) and nanoplastic particles (NPPs) in the absence and presence of humic acids (HAs), an important environmental factor in aquatic systems. Four stress response and detoxification genes (CAT, GST, HSP70, and P-GP) were used to characterize the toxic response of the neonates to the exposure. Our results showed that NPPs were much more toxic than MPPs in that 10 mg L−1 NPPs induced over 70% of death in 96 h but MPPs (as high as 400 mg L−1) caused no mortality under all tested conditions. More importantly, we revealed a potent protective role of HA against NPP toxicity at environmentally relevant concentrations. The effect was concentration dependent, as 50 mg L−1 HA subdued the NPP (400 mg L−1) toxicity effect completely. NPPs elicited the up-regulation of all examined genes while HA diminished the change appreciably, further confirming its detoxifying role against NPP toxicity. Through fluorescence and dynamic light scattering measurements, we found that HA was adsorbed on NPPs and formed a corona, without causing agglomeration or precipitation, but changed NPP distribution in the D. magna neonates in a way similar to that of MPPs, leading to alleviated toxicity. Our results suggest that it is essential to consider environmental factors in evaluating and monitoring NPP toxicity as this presents a more relevant exposure state.

Chlorinated polyfluoroalkylether sulfonates (Cl-PFAESs) have been used as perfluorooctanesulfonate (PFOS) alternatives in the chrome plating industry for years. Although Cl-PFAESs have become ubiquitous environmental contaminants, knowledge on their toxicological mechanism remains very limited. We compared potential thyroid hormone (TH) disruption effects of Cl-PFAESs and PFOS via the mechanisms of competitive binding to TH transport proteins and activation of TH receptors (TRs). Fluorescence binding assays revealed that 6:2 Cl-PFAES, 8:2 Cl-PFAES and F-53B (a mixture of 6:2 and 8:2 Cl-PFAES) all interacted with a TH transport protein transthyretin (TTR), with 6:2 Cl-PFAES showing the highest affinity. It was also found that the chemicals interacted with TRs, with the affinity following the order of 6:2 Cl-PFAES > PFOS > 8:2 Cl-PFAES. In reporter gene assays the chemicals exhibited agonistic activity toward TRs, with the potency of 6:2 Cl-PFAES comparable to that of PFOS. The chemicals also promoted GH3 cell proliferation, with 6:2 Cl-PFAES displaying the highest potency. Molecular docking and molecular dynamic simulation revealed that both Cl-PFAESs fit into the ligand binding pockets of TTR and TRs with the binding modes similar to PFOS. Collectively, our results demonstrate that Cl-PFAESs might cause TH disruption effects through competitive binding to transport proteins and activation of TRs.

// Tianyi Dong 1, 2, * , Yanping Peng 1, * , Ning Zhong 3 , Fengyan Liu 3 , Hanyu Zhang 1 , Mengchen Xu 4 , Rutao Liu 4 , Mingyong Han 5 , Xingsong Tian 2 , Jihui Jia 1 , Lap Kam Chang 1 , Liang-Hong Guo 6 and Shili Liu 1 1 School of Medicine, Shandong University, Jinan, Shandong, 250012, China 2 Department of Breast Thyroid Surgery, Shandong Provincial Hospital, Shandong University, Jinan, Shandong, 250021, China 3 Department of Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong, 250012, China 4 School of Environmental Science and Engineering, Shandong University, Jinan, Shandong, 250100, China 5 Cancer Therapy and Research Center, Shandong Provincial Hospital, Shandong university, Jinan, Shandong 250021, China 6 State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China * These authors contributed equally to this work Correspondence to: Liang-Hong Guo, email: lhguo@rcees.ac.cn Shili Liu, email: liushili@sdu.edu.cn Keywords: PFDA, proliferation, sPLA2-IIA, TCF4 Received: September 06, 2016 Accepted: April 05, 2017 Published: April 20, 2017 ABSTRACT The association of perfluorodecanoicacid (PFDA) with tumor promotion and associated effects is not clear. Given that PDFA is mostly consumed with food and drinking water, we evaluated the effects of PFDA on a gastric cell line. When added to cell cultures, PFDA significantly increased growth rate and colony forming ability compared with control treatment. We found that suppression of cell senescence, but not apoptosis or autophagy was associated with PFDA-induced promotion of cell amount. To determine the molecular mechanism that was involved, DNA microarray assays was used to analyze changes in gene expression in response to PFDA treatment. Data analysis demonstrated that the vascular endothelial growth factor signaling pathway had the lowest p -value, with sPLA2-IIA ( pla2g2a ) exhibits the most altered expression pattern within the pathway. Moreover, sPLA2-IIA and its transcription factor TCF4, known as a direct target and a binding partner of Wnt/β-catenin signaling in gastric cells respectively, were the third and second most varied genes globally. Cells transfected with expression plasmids pENTER- tcf4 and pENTER- pla2g2a show reduced cell proliferation by more than 60% and 30% respectively. Knockdown with sPLA2-IIA siRNA provided additional evidence that sPLA2-IIA was a mediator of PFDA-induced cell senescence suppression. The results suggest for the first time that PFDA induced suppression of cell senescence through inhibition of sPLA2-IIA protein expression and might increased the proliferative capacity of an existing tumor.

Chlorinated polyfluoroalkylether sulfonic acids (Cl-PFESAs) have been shown to have potential thyroid hormone (TH) disruption effects. Here, we further investigated their estrogenic effects and underlying mechanisms. In vivo results revealed that exposure of zebrafish to Cl-PFESAs induced disorder of sex hormones during the early embryonic stages and caused histopathological lesions in the gonads of adult zebrafish relative to control groups. To find out whether the estrogen receptor is the molecular target of Cl-PFESAs, the binding interaction between Cl-PFESAs and ERs was investigated using a series of in vitro assays. We found that all tested chemicals could bind directly to ERs and exhibit relatively weak agonistic activity toward ERs, suggesting that the ER-mediated signaling pathway is directly involved in the estrogenic effects of Cl-PFESAs. The internal dose of 8:2 Cl-PFESA was significantly higher than the others, which explained why it obviously displayed an ER agonistic effect despite its weak ER binding affinity. Taken together, these results uncover that, in addition to the TH disruption effect, Cl-PFESAs might also cause estrogenic effects by activating ER pathways.

Carbon nanomaterials (CNMs) are widely used in industrial and medical sectors. The increasing exposure of CNMs necessitates the studies of their potential environmental and health effects. High-mobility group box-1 (HMGB1) is a nuclear DNA-binding protein, but when released from cells, may cause sustained inflammatory response and promote cell migration and invasion. In this work, we found that 7-day exposure of 2.5 mg/kg/day CNMs, including C60, single-walled carbon nanotubes, and graphene oxides significantly elevated the level of HMGB1 in blood and lung lavage fluids in C57BL/6 mice. Subsequently, cellular effects and underlying mechanism were explored by using Raw264.7. The results showed that noncytotoxic CNMs enhanced HMGB1 intracellular translocation and release via activating P2X7 receptor. Released HMGB1 further activated receptor for advanced glycation endproducts (RAGE) and downstream signaling pathway by upregulating RAGE and Rac1 expression. Simultaneously, CNMs prepared the cells for migration and invasion by modulating MMP2 and TIMP2 gene expression as well as cytoskeleton reorganization. Intriguingly, released HMGB1 from macrophages promoted the migration of nearby lung cancer cell, which can be efficiently inhibited by neutralizing antibodies against HMGB1 and RAGE. Taken together, our work demonstrated that CNMs stimulated HMGB1 release and cell migration/invasion through P2X7R-HMGB1-RAGE pathway. The revealed mechanisms might facilitate a better understanding on the inflammatory property and subsequent cell functional alteration of CNMs.

Polybrominated diphenyl ethers (PBDEs) can be metabolized to hydroxylated PBDEs (OH-PBDEs), which play important roles in their disruption effects on the thyroid hormone (TH) system. Recently, multiple in vitro studies suggested that OH-PBDEs might be further metabolically transformed to PBDE sulfates. However, information about the bioactivity of PBDE sulfate metabolites is limited. In the present study, we explored the possible disruption effects of PBDE sulfates to the TH system by studying their binding and activity towards TH transport proteins and nuclear receptors. We found PBDE sulfates could bind to two major TH transport proteins (thyroxine-binding globulin and transthyretin). Besides, PBDE sulfates could also bind to two subtypes of TH nuclear receptors (TRα and TRβ) and showed agonistic activity towards the subsequent signaling pathway. Moreover, the PBDE sulfates showed higher binding potency to TH transport proteins and TRs compared with their corresponding OH-PBDE precursors. Molecular docking results showed that replacement of hydroxyl groups with sulfate groups might lead to more hydrogen bond interactions with these proteins. Overall, our study suggested that PBDE sulfates might disturb the TH system by binding to TH transport proteins or TRs. Our finding indicated a possible mechanism for the TH system disruption effects of PBDEs through their sulfate metabolites.

Biotransformation of 8:2 fluorotelomer alcohol (8:2 FTOH) can form potentially more toxic metabolites. However, the responsible cytochrome P450 (CYP) isoform(s) and phase II metabolism have not been studied in humans. Here, we characterized the in vitro metabolism of 8:2 FTOH by recombinant human CYPs, human liver microsomes, and human liver cytosol. The results showed that among the 11 isoforms investigated, CYP2C19 was the only enzyme capable of catalyzing 8:2 FTOH with Km and Vmax values of 18.8 μM and 8.52 pmol min−1 pmol−1 P450, respectively. The phase I metabolite was identified as 8:2 fluorotelomer aldehyde (8:2 FTAL). HLMs also catalyzed 8:2 FTOH transformation, with the Vmax and intrinsic clearance (CLint) values similar to those of CYP2C19 after the protein content is taken into account. Molecular docking showed that the hydroxyl group of 8:2 FTOH accesses the heme iron-oxo of CYP2C19 in an energetically favored orientation. 8:2 FTOH was also transformed by phase II enzymes to form O-glucuronide and O-sulfate conjugates. The CLint values follow the order of sulfation > oxidation > glucuronidation, suggesting that conjugation is the major metabolic pathway, which explains the low yield of perfluoroalkyl acids (PFCAs). These results provide new insight into fluorotelomer alcohol biotransformation and indirect human exposure to PFCAs.

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that is used worldwide and is continuously being detected in biota and the environment, thus presenting potential threats to the ecosystem and human health. Although PFOS is highly immunotoxic, its underlying molecular mechanisms remain largely unknown. The present study examined PFOS-induced immunotoxicity in the mouse spleen and explored its underlying mechanisms by gene expression profiling. Oral exposure of male BALB/c mice for three weeks followed by one-week recovery showed that a 10 mg/kg/day PFOS exposure damaged the splenic architecture, inhibited T-cell proliferation in response to mitogen, and increased the percentages of T helper (CD3(+)CD4(+)) and cytotoxic T (CD3(+)CD8(+)) cells, despite the decrease in the absolute number of these cells. A delayed type of PFOS immunotoxicity was observed, which mainly occurred during the recovery period. Global gene expression profiling of mouse spleens and QRT-PCR analyses suggest that PFOS inhibited the expression of genes involved in cell cycle regulation and NRF2-mediated oxidative stress response, and upregulated those in TCR signaling, calcium signaling, and p38/MAPK signaling pathways. Western blot analysis confirmed that the expressions of CAMK4, THEMIS, and CD3G, which were involved in the upregulated pathways, were induced upon PFOS exposure. Acute PFOS exposure modulated calcium homoeostasis in splenocytes. These results indicate that PFOS exposure can activate TCR signaling and calcium ion influx, which provides a clue for the potential mechanism of PFOS immunotoxicity. The altered signaling pathways by PFOS treatment as revealed in the present study might facilitate in better understanding PFOS immunotoxicity and explain the association between immune disease and PFOS exposure.

Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and are often detected in the environment, wildlife, and humans, presenting potential threats to ecosystem and human health. PBDEs can cause neurotoxicity, hepatotoxicity, and endocrine disruption. However, data on PBDE immunotoxicity are limited, and the toxicity mechanisms remain largely unknown. Both immune cell death and dysfunction can modulate the responses of the immune system. This study examined the toxic effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and decabromodiphenyl ether (BDE-209) on the immune system by using peritoneal macrophages as the model. The macrophages were exposed to PBDEs, and cell death was determined through flow cytometry and immunochemical blot. The results showed that after 24h of exposure, BDE-47 (>5 μM) and BDE-209 (>20 μM) induced cell apoptosis, increased intracellular reactive oxygen species (ROS) formation and depleted glutathione. BDE-47 was more potent than BDE-209; the cytotoxic concentrations for BDE-47 and BDE-209 were determined to be 5 μM and 20 μM, respectively, during 24h of exposure. However, pretreatment with n-acetyl-l-cysteine (ROS scavenger) partially reversed the cytotoxic effects. Further gene expression analyses on Caspase-3,-8,-9, TNFR1, and Bax revealed that both intrinsic and extrinsic apoptotic pathways were activated. More importantly, non-cytotoxic concentrations BDE-47 (

The noncovalent interactions of 24 diversely structured hydroxylated polybrominated diphenyl ethers (OH-PBDEs), ranging from hydroxylated monobromodiphenyl ether (OH-monoBDE) to hydroxylated octabromodiphenyl ether (OH-octaBDE), with bovine serum albumin (BSA) have been examined by employing an electrospray ionization source fitted on a quadrupole time-of-flight hybrid mass spectrometer equipped with a traveling wave ion mobility cell. The mass spectrometric parameters were finely optimized to favor the observation of noncovalent complexes. The experimental data confirm that due to the close structural resemblance with thyroid hormone thyroxine, some OH-PBDEs have been shown to conjugate with BSA. It is found that BSA preferentially interacts with one OH-octaBDE (4′-OH-BDE-201) and two hydroxylated heptabromodiphenyl ether (OH-heptaBDE) isomers (4-OH-BDE-187 and 6-OH-BDE-180) with 1:1 and 2:1 binding stoichiometries. The state of bound OH-PBDEs is inferred to be as both neutral and anionic species. The dissociation constants of corresponding noncovalent complexes are calculated by titration curve data fitting. Investigation into various OH-PBDE analogs demonstrates that both the degree of bromination and the positions of hydroxylation and bromine moieties may exert influence on the binding of OH-PBDEs to BSA. The possible conformational changes in BSA induced upon the binding of OH-PBDEs were explored by use of ion mobility-mass spectrometry (IM-MS) to understand the details of the interactions in structural aspects.

Quantitative detection of small molecules by a photoelectrochemical system was demonstrated in a competitive assay for biotin. The system for the determination of Biotin was employed by using ruthenium tris(bipyridine) (Ru-bpy) as the label, oxalate as the sacrificial electron donor to recycle the label, and tin oxide nanoparticle as the semiconductor electrode. Upon irradiation with 470 nm light, electrons of Ru-bpy were promoted to the excited state, and then injected into the conduction band of the tin oxide semiconductor, thus producing photocurrent signal. The oxidized Ru-bpy was reduced back to its original state by oxalate in solution, and was used again in the next cycle of signal generation. In the competitive assay of biotin, avidin was adsorbed passively on the tin oxide surface as the recognition element. Adsorbed protein was found to be stable under assay conditions, and reached maximum surface coverage when the concentration of avidin solution for adsorption was 0.5 g L −1 or higher. A mixed solution of 1 μM tracer and various concentrations of biotins were reacted with surface-immobilized avidin. As biotin concentration was increased, less tracer molecules bound to avidin, leading to a reduction in its photocurrent signal. A detection limit of 8 μg L −1 biotin was obtained in the competitive assay. The method can be easily extended to the detection of competitive immunoassays for organic chemicals.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) plays a critical role in modulating protein function in many cellular processes and human diseases such as Alzheimer's disease and type II diabetes, and has emerged as a promising new target. Specific inhibitors of OGT could be valuable tools to probe the biological functions of O-GlcNAcylation, but a lack of robust nonradiometric assay strategies to detect glycosylation, has impeded efforts to identify such compounds. Here we have developed a novel label-free electrochemical biosensor for the detection of peptide O-GlcNAcylation using protease-protection strategy and electrocatalytic oxidation of tyrosine mediated by osmium bipyridine as a signal reporter. There is a large difference in the abilities of proteolysis of the glycosylated and the unglycosylated peptides by protease, thus providing a sensing mechanism for OGT activity. When the O-GlcNAcylation is achieved, the glycosylated peptides cannot be cleaved by proteinase K and result in a high current response on indium tin oxide (ITO) electrode. However, when the O-GlcNAcylation is successfully inhibited using a small molecule, the unglycosylated peptides can be cleaved easily and lead to low current signal. Peptide O-GlcNAcylation reaction was performed in the presence of a well-defined small-molecule OGT inhibitor. The results indicated that the biosensor could be used to screen the OGT inhibitors effectively. Our label-free electrochemical method is a promising candidate for protein glycosylation pathway research in screening small-molecule inhibitors of OGT.

Perfluoroalkyl substances (PFASs) have been shown to cause abnormal levels of thyroid hormones (THs) in experimental animals, but the molecular mechanism is poorly understood. Here, a fluorescence displacement assay was used to determine the binding affinities of 16 PFASs with two major TH transport proteins, transthyretin (TTR) and thyroxine-binding globulin (TBG). Most of the tested PFASs bound TTR with relative potency (RP) values of 3×10(-4) to 0.24 when compared with that of the natural ligand thyroxine, whereas fluorotelomer alcohols did not bind. Only perfluorotridecanoic acid and perfluorotetradecanoic acid bound TBG, with RP values of 2×10(-4) when compared with that of thyroxine. Based on these results, it was estimated that displacement of T4 from TTR by perfluorooctane sulfonate and perfluorooctanoic acids would be significant for the occupationally exposed workers but not the general population. Structure-binding analysis revealed that PFASs with a medium chain length and a sulfonate acid group are optimal for TTR binding, and PFASs with lengths longer than 12 carbons are optimal for TBG binding. Three mutant proteins were prepared to examine crucial residues involved in the binding of PFASs to TH transport proteins. TTR with a K15G mutation and TBG with either a R378G or R381G mutation showed decreased binding affinity to PFASs, indicating that these residues play key roles in the interaction with the compounds. Molecular docking showed that the PFASs bind to TTR with their acid group forming a hydrogen bond with K15 and the hydrophobic chain towards the interior. PFASs were modeled to bind TBG with their acid group forming a hydrogen bond with R381 and the hydrophobic chain extending towards R378. The findings aid our understanding of the behavior and toxicity of PFASs on the thyroid hormone system.

Perfluoroalkyl acids (PFAAs) are widespread environmental contaminants which have been detected in humans and linked to adverse health effects. Previous toxicological studies mostly focused on nuclear receptor-mediated pathways and did not support the observed toxic effects. In this study, we aimed to investigate the molecular mechanisms of PFAA toxicities by identifying their biological targets in cells. Using a novel electrochemical biosensor, 16 PFAAs were evaluated for inhibition of protein tyrosine phosphatase SHP-2 activity. Their potency increased with PFAA chain length, with perfluorooctadecanoic acid (PFODA) showing the strongest inhibition. Three selected PFAAs, 25 μM perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid, and PFODA, also inhibited SHP-2 activity in HepG2 cells and increased paxillin phosphorylation level. PFOA was detected in the immunoprecipitated SHP-2 from the cells exposed to 250 μM PFOA, providing unequivocal evidence for the direct binding of PFOA with SHP-2 in the cell. Molecular docking rationalized the formation of PFAA/SHP-2 complex and chain length-dependent inhibition potency. Our results have established SHP-2 as a new cellular target of PFAAs.

Four ELISA formats, antigen-coated indirect, antigen-coated direct, antibody-coated, and the secondary antibody-coated, were developed using monoclonal antibody to determine cephalexin. Results showed that the secondary antibody-coated method of ELISA had a better performance in the establishment of standard curves. The optimized secondary antibody-coated ELISA was used to determine cephalexin spiked in pig muscle, pig kidney, pig liver, chicken muscle, chicken liver, and cow's milk. The limits of detection were 0.09 ng/g, 0.15 ng/g, 0.26 ng/g, 0.13 ng/g, 0.19 ng/g, and 0.08 ng/mL in pig muscle, pig kidney, pig liver, chicken muscle chicken liver, and cow's milk, respectively. A mean recovery of 77.2–128.5% and coefficient of variation of 2.6–14.7% were obtained. The results given by the ELISA method were in agreement with those of the LC-MS/MS method, which confirmed the potential of the ELISA method for the monitoring of cephalexin in milk and animal tissues.

An indirect competitive fluorescence immunoassay using a DNA/dye conjugate as antibody multiple labels was developed on 96-well plates for the identification and quantification of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in aqueous samples. A hapten, 2,4,2'-tribromodiphenyl ether-4'-aldehyde, was synthesized, and was conjugated to bovine serum albumin to form a coating antigen. Specific recognition of the antigen by anti-PBDE antiserum was confirmed by a surface plasmon resonance measurement. In the immunoassay, the coating antigen was adsorbed on a 96-well plate first, and a sample, antiserum and biotinylated goat anti-rabbit secondary antibody were then added and reacted sequentially. A biotinylated, double-stranded DNA with 219 base pairs was attached to the secondary antibody by using streptavidin as a molecular bridge. In situ multiple labeling of the antibody was accomplished after addition of a DNA-binding fluorescent dye, SYBR Green I. The working range of the immunoassay for the BDE-47 standard was 3.1-390 microg/L, with an IC50 value of 15.6 microg/L. The calculated LOD of the immunoassay is 0.73 microg/L. The immunoassay demonstrated relatively high selectivity for BDE-47, showing very low cross-reactivity (< 3%) with BDE-15, BDE-153 and BDE-209. With a spiked river water sample containing 50 microg/L BDE-47, quantification by the immunoassay was 41.9 microg/L, which compared well with the standard GC-ECD method (45.7 microg/L). The developed immunoassay provides a rapid screening tool for polybrominated diphenyl ethers in environmental samples.

A novel label-free electrochemical method for measuring the activity of protein tyrosine kinases (PTK) has been developed. Epidermal growth factor receptor (EGFR), a typical PTK associated with a large percentage of all solid tumors, was used as the model kinase. Poly(glu, tyr) (4:1) peptide, as a substrate of EGFR, was covalently immobilized on the surface of indium tin oxide (ITO) electrode by silane chemistry. The tyrosine (Tyr) residue in the polypeptide served as an electrochemical signal reporter. Its voltammetric current was catalyzed by a dissolved electron mediator Os(bpy) 3 2+ (bpy = 2,2′-bipyridine) for increased sensitivity. Phosphorylation of the Tyr led to a loss of its electrochemical current, thus providing a sensing mechanism for PTK activity. Experimental conditions for the silanization of ITO surface and immobilization of polypeptide were investigated in details to facilitate the generation of Tyr electrochemical signal. The proposed biosensor exhibited high sensitivity and excellent stability. The limit of detection for EGFR was 1 U mL −1 . Furthermore, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent electrochemical signal, the half-maximal inhibition value IC 50 of three EGFR inhibitors, PD-153035, OSI-774 and ZD-1839, and their corresponding inhibition constants K i were estimated, which were in agreement with those obtained from the conventional kinase assay. This electrochemical biosensor can be implemented in an array format for the high throughput assay of in vitro PTK activity and PTK inhibitors screening for practical diagnostic application and drug discovery.

Silica nanoparticles are most commonly modified with amino-silanes, followed by post-modification activation for protein immobilization. In this work, epoxy-functionalized silica nanoparticles were prepared by modification with glycidyloxypropyl trimethoxysilane (GPTMS) for direct protein immobilization. Silica nanoparticles possessed an average size of 46 nm, but increased to 63 nm after GPTMS modification. Reaction time, reaction temperature and GPTMS content had no significant effect on particle size. Zeta potential of SiO2 changed from −26mV to +38mV after modification. Fourier-transformed infrared spectroscopy revealed alkyl C-H bending and stretching bands at 2944 cm−1, 1343 cm−1 and 1465 cm−1, respectively, for the modified nanoparticles. Fluorescein cadaverine was found to bind to GPTMS-modified SiO2, but not to bare SiO2, indicating the chemical reactivity of epoxy groups on the modified nanoparticle with amines. Finally, fluorescently labeled bovine serum albumin (BSA) was used as a model protein to investigate the capacity of epoxy-SiO2 nanoparticles for protein immobilization. The results showed that more proteins were immobilized on the particle with longer reaction time, higher NaCl concentration, lower pH, and less GPTMS content. More importantly, proteins bound to epoxy-SiO2 nanoparticle were highly stable. Under optimized reaction conditions, as much as 25 mg BSA/g nanoparticle was covalently attached to the nanoparticle. The epoxy silane modification of silica nanoparticles offers a reactive surface for one-step and high-density protein immobilization.

Facile electrical communication between redox-active labeling molecules and electrode is essential in the electrochemical detection of bio-affinity reactions. In this report, nanometer-sized indium tin oxide (ITO) particles were employed in the fabrication of porous thick film electrodes to enhance the otherwise impeded electrochemical activity of redox labels in multi-layered protein films, and to enable quantitative detection of avidin/biotin binding interaction. To carry out the affinity reaction, avidin immobilized on an ITO electrode was reacted with mouse IgG labeled with both biotin and ruthenium Tris-(2.2'-bipyridine) (Ru-bipy). The binding reaction between avidin and biotin was detected by the catalytic voltammetry of Ru-bipy in an oxalate- containing electrolyte. On sputtered ITO thin film electrode, although a single layer of Ru-bipy labeled avidin exhibited substantial anodic current, attaching the label to the outer IgG layer of the avidin/biotin-IgG binding pair resulted in almost complete loss of the signal. However, electrochemical current was recovered on ITO film electrodes prepared from nanometer-sized particles. The surface of the nanoparticle structured electrode was found by scanning electron microscopy to be very porous, and had twice as much surface binding capacity for avidin as the sputtered electrode. The results were rationalized by the assumption of different packing density of avidin inner layer on the two surfaces. and consequently different electron transfer distance between the electrode and Ru-bipy on the IgG outer layer. A linear relationship between electrochemical current and IgG concentration was obtained in the range of 40-4000 nmol L(-1) on the nanoparticle-based electrode. The approach can be employed in the electrochemical detection of immunoassays using non-enzymatic redox labels. (C) 2007 Elsevier B.V. All rights reserved.

Human G protein-coupled receptor 40 (hGPR40), with medium- and long-chain free fatty acids (FFAs) as its natural ligands, plays an important role in the enhancement of glucose-dependent insulin secretion. To date, information about the direct binding of FFAs to hGPR40 is very limited, and how carbon-chain length affects the activities of FFAs on hGPR40 is not yet understood. In this study, a fluorescein-fasiglifam analogue (F-TAK-875A) conjugate was designed and synthesized as a site-specific fluorescence probe to study the interaction of FFAs with hGPR40. hGPR40 was expressed in human embryonic kidney 293 cells and labeled with F-TAK-875A. By using flow cytometry, competitive binding of FFA and F-TAK-875A to hGPR40-expressed cells was measured. Binding affinities of 18 saturated FFAs, with carbon-chain lengths ranging from C6 to C23, were analyzed. The results showed that the binding potencies of FFAs to hGPR40 were dependent on carbon length. There was a positive correlation between length and binding potency for seven FFAs (C9-C15), with myristic acid (C15) showing the highest potency, 0.2% relative to TAK-875. For FFAs with a length of fewer than C9 or more than C15, they had very weak or no binding. Molecular docking results showed that the binding pocket of TAK-875 in hGPR40 could enclose FFAs with lengths of C15 or fewer. However, for FFAs with lengths longer than C15, part of the alkyl chain extended out of the binding pocket. This study provided insights into the structural dependence of FFAs binding to and activation of hGPR40.

Previous animal experiments have implied that organophosphate esters (OPEs) have a disruption effect on the thyroid endocrine system. However, knowledge of the toxicological mechanism remains limited. In this study, the activities of four OPEs have been characterized against the thyroid hormone (TH) nuclear receptor (TR) using two in vitro models, with the aim of evaluating their toxicity mechanisms towards the TR. The results of a TH-dependent cell proliferation assay showed that tris(2-chloro-1-(chloromethyl)ethyl)phosphate (TDCPP) could induce cell growth, while the other three OPEs had no effect. The results of a luciferase reporter gene assay revealed that all four of the OPEs tested in the current study showed agonistic activity towards TRβ, with TDCPP being the most potent one. Moreover, molecular docking revealed that all the tested OPEs could fit into the ligand binding pocket of TRβ, with TDCPP binding more effectively than the other three OPEs. Taken together, these data suggest that OPEs might disrupt the thyroid endocrine system via a mechanism involving the activation of TR.

A novel and robust amperometric enzyme electrode for the detection of glucose has been constructed by immobilizing glucose oxidase (GOD) on a boron-doped diamond (BDD) electrode with a cross-linking technique. Cyclic voltammograms were used to characterize the enzyme electrode. The response was evaluated with respect to the enzyme amount on the electrode and the concentration of BSA used in the cross-linking reagent. Linear calibration curve was obtained for glucose over the concentration range up to 35 mM in phosphate buffer, with the lowest experimental value measured being 0.5 mM.

A new electrochemiluminescence (ECL) sensor was developed for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) quantification and Escherichia coil formamidopyrimidine-DNA glycosylase (FPG) activity assay. The sensor employed a novel spermine conjugated ruthenium tris-(bipyridine) derivative (spermine-Ru) which binds specifically with 8-oxodGuo through a one-step reaction and also acts as an ECL signal reporter. In the sensor, an 8-oxodGuo-containing ds-DNA film was first immobilized on a gold electrode by self-assembly. The DNA film was then incubated with spermine-Ru under oxidative condition for 8-oxodGuo labeling. The ECL intensity was found to correlate with the amount of 8-oxodGuo on the surface and the detection limit was estimated to be about 1 lesion in 500 DNA bases. Addition of FPG resulted in some loss of the signal due to the excision of 8-oxodGuo by the enzyme. An inverse relationship between ECL intensity and FPG concentration was observed in a range from 0 to 4.0 U/mu L, demonstrating that this sensor could be used for FPG activity assay. A number of metal ions were screened by the sensor for their inhibition effect on FPG activity. Among them, Hg2+ and methyl Hg(II) shown very potent inhibition, with IC50 values of 4.04 mu M and 4.34 nM respectively. The result may suggest that interference on the DNA repair system could be another mechanism for the high toxicity of MeHg. (C) 2015 Elsevier B.V. All rights reserved.

Perfluoroalkyl adds (PFAAs) have been shown to bind with hepatic peroxisome proliferator receptor a, estrogen receptors and human serum albumin and subsequently cause some toxic effects. Lysine decarboxylase (LDC) plays an important role in cell growth and developmental processes. In this study, the inhibitory effect of 16 PFAAs, including 13 perfluorinated carboxylic acids (PFCAs) and 3 perfluorinated sulfonic acids (PFSAs), on lysine decarboxylase (LDC) activity was investigated. The inhibition constants obtained in fluorescence enzyme assays fall in the range of 2.960 mu pM to 290.8 mu M for targeted PFCAs, and 41.22 mu M to 67.44 mu M for targeted PFSAs. The inhibitory effect of PFCAs increased significantly with carbon chain (7-18 carbons), whereas the short chain PFCAs (less than 7 carbons) did not show any effect. Circular dichroism results showed that PFAA binding induced significant protein secondary structural changes. Molecular docking revealed that the inhibitory effect could be rationalized well by the cleft binding mode as well as the size, substituent group and hydrophobic characteristics of the PFAAs. At non-cytotoxic concentrations, three selected PFAAs inhibited LDC activity in HepG2 cells, and subsequently resulted in the decreased cadaverine level in the exposed cells, suggesting that LDC may be a possible target of PFAAs for their in vivo toxic effects. (C) 2014 Elsevier B.V. All rights reserved.

Polybrominated diphenyl ethers (PBDEs) have been shown to alter thyroid hormone level in experimental animals. One of the possible mechanisms for hormone disruption is the competitive binding of hydroxylated PBDEs (OH-PBDEs) with hormone transport proteins. In this study, binding interaction of 14 diversely structured OH-PBDEs with two thyroxine transport proteins was investigated by fluorescence displacement assay, circular dichroism, and molecular docking. Binding affinity of the 14 OH-PBDEs with transthyretin (TTR) and thyroxine-binding globulin (TBG) was measured by competitive fluorescence displacement assay. The binding constant was found to fall in the range of 1.4 x 10⁷ M and 6.9 x 10⁸ M⁻¹ for TTR, and between 6.5 x 10⁶ M⁻¹ and 2.2 x 10⁸ M⁻¹ for TBG. Binding affinity increased significantly with bromination number from 1 to 4, whereas 5- and 6-brominated diphenyl ethers did not show any further increase. Protein secondary structural change of TTR and TBG upon binding with 5-OH-BDE-047 was investigated by circular dichroism. The spectral change displayed a pattern similar to the one with thyroxine, suggesting that the environmental chemical binds to the two proteins at the same sites as the hormone. In molecular docking analysis, a ligand-binding channel in TTR was revealed for OH-PBDEs binding, which appeared to be mostly hydrophobic inside but guarded by positively charged residue Lys15 at the entrance. Binding affinity of the 14 OH-PBDEs with TTR could be rationalized reasonably well by their pocket binding mode and hydrophobic characteristics. Based on the binding constant obtained in this work, possibility of in vitro competitive displacement of thyroid hormones from the transport proteins by OH-PBDEs was evaluated.

Direct electrochemistry of site-specific mutants of yeast iso-1-cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge-plane pyrolytic graphite (EPG) and bis(4-pyridyl)-disulphide-modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a variety of electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile-Cys102Thr greater than Cys102Thr greater than Asn52Ala-Cys102Thr. Mid-point potentials follow the order: Arg13Ile (+60 +/- 5 mV vs. standard calomel electrode) greater than Cys102Thr (+40 +/- 5 mV) greater than Lys27Gln (+30 +/- 5 mV) approximately Lys72Asp (+30 +/- 5 mV) greater than Asn52Ala-Cys102Thr (+15 +/- 5 mV) greater than Asn52Ile-Cys102Thr (-10 +/- 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical response are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single-site variation could modify or abolish direct electrochemistry in the ternary adduct.

Binding of five perfluoroalkyl acids with human serum albumin (HSA) was investigated by site-specific fluorescence. Intrinsic fluorescence of tryptophan-214 in HSA was monitored upon addition of the chemicals. Although perfluorobutyl acid (PFBA) and perfluorobutane sulfonate (PFBS) did not cause fluorescence change, perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorododecanoic acid (PFDoA) induced fluorescence quenching, from which binding constant of 2.7 x 10(5) M(-1) for PFOA and 2.2 x 10(4) M(-1) for PFOS was calculated. Two fluorescent probes, dansylamide (DA) and dansyl-L: -proline (DP), were employed in fluorescence displacement measurements to study the interaction at two Sudlow's binding sites. At Site I, both PFBA and PFBS displaced DA with binding constants of 1.0 x 10(6) M(-1) and 2.2 x 10(6) M(-1). At Site II, PFBS and PFDoA displaced DP with binding constants of 6.5 x 10(6) M(-1) and 1.2 x 10(6) M(-1), whereas PFBA did not bind. The data were compared with fatty acids to evaluate the potential toxicological effect of these environmental chemicals.

Photoelectrochemical sensors were developed for the rapid detection of oxidative DNA damage induced by Fe2+ and H2O2 generated in situ by the enzyme glucose oxidase. The sensor is a multilayer film prepared on a tin oxide nanoparticle electrode by layer-by-layer self-assembly and is composed of separate layers of a photoelectrochemical indicator, DNA, and glucose oxidase. The enzyme catalyzes the formation of H2O2 in the presence of glucose, which then reacts with Fe2+ and generates hydroxyl radicals by the Fenton reaction. The radicals attack DNA in the sensor film, mimicking metal toxicity pathways in vivo. The DNA damage is detected by monitoring the change of photocurrent of the indicator. In one sensor configuration, a DNA intercalator, Ru(bpy)2(dppz)2+ (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine), was employed as the photoelectrochemical indicator. The damaged DNA on the sensor bound less Ru(bpy)2(dppz)2+ than the intact DNA, resulting in a drop in photocurrent. In another configuration, ruthenium tris(bipyridine) was used as the indicator and was immobilized on the electrode underneath the DNA layer. After oxidative damage, the DNA bases became more accessible to photoelectrochemical oxidation than the intact DNA, producing a rise in photocurrent. Both sensors displayed substantial photocurrent change after incubation in Fe2+/glucose in a time-dependent manner. And the detection limit of the first sensor was less than 50 microM. The results were verified independently by fluorescence and gel electrophoresis experiments. When fully integrated with cell-mimicking components, the photoelectrochemical DNA sensor has the potential to become a rapid, high-throughput, and inexpensive screening tool for chemical genotoxicity.

The direct electrochemistry of the cytochrome c/cytochrome b5 and cytochrome c/plastocyanin complexes has been investigated at edge-plane graphite and modified gold electrode surfaces, which are selective for one of the two components of the complex. Electrochemical response of one protein at an otherwise electrostatically unfavorable electrode surface was achieved in the presence of the other protein, and the calculated heterogeneous electron-transfer rate constant and diffusion coefficient were found to be in good agreement with the values determined previously from the electrochemistry of the individual proteins [Armstrong, F. A., Hill, H. A. O., & Walton, N. J. (1988) Acc. Chem. Res. 21, 407 and references therein]. A dynamic model of the protein-protein-electrode ternary complex is proposed to explain the promotion effect, and this model is supported by a study comparing the electrochemical responses of covalent and electrostatic cytochrome c/plastocyanin complexes. It is also suggested that the behavior of protein-protein complexes at electrode surfaces could be related to that of the complexes associated with biological membranes.

The electrochemistry of the enzyme, sulfide:cytochrome c oxidoreductase, also known as flavocytochrome c552 from the purple sulfur bacterium, Chromatium vinosum, has been studied using several modified electrodes. Direct electron transfer between the heme of the flavocytochrome and an electrode is observed in the presence of a redox-inactive cationic species which promotes the voltammetry of the enzyme. Quasi-reversible electron transfer was achieved using the aminoglycoside, neomycin, as a promoter at either a modified gold or polished edge-plane graphite electrode. Further evidence for direct electron transfer is provided by the catalytic response of the enzyme at the electrode in the presence of substrate. Also reported is the direct spectroelectrochemistry of flavocytochrome c552 at an optically transparent thin layer gold electrode modified with Cys-Glu-Cys in the presence of neomycin.

Rapid and sensitive detection methods are in urgent demand for the screening of an overwhelming number of existing and new chemicals as potential DNA-damaging agents. In this study, two photoelectrochemistry-based DNA sensor configurations were employed in the detection of DNA damage caused by styrene oxide and Fe2+/H2O2. The organic compound and heavy metal represent genotoxic chemicals possessing two major damaging mechanisms, DNA adduct formation and DNA oxidation. In the first sensor configuration, a ruthenium tris(bipyridine)-labeled avidin film and a double-stranded calf thymus DNA (ds-DNA) film were assembled successively on tin oxide nanoparticle film electrodes. Photogenerated Ru(III) oxidized guanidine and adenosine bases in DNA and gave rise to photocurrent. DNA damage was detected after the reaction of the DNA film with either styrene oxide or Fe2+/H2O2, which exposed more DNA bases for photooxidation and resulted in increased photocurrent. In the second configuration, an unlabeled avidin film and a ds-DNA film were assembled on the semiconductor electrode. A DNA intercalator, Ru(bpy)2(dppz)2+ (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a: 2',3'-c]phenazine), was employed as the photoelectrochemical signal reporter. After the chemical reaction with the damaging agents, the DNA film bound less Ru(bpy)2(dppz)2+, accompanied by a drop in photocurrent. Both sensors were used to follow the reaction course in styrene oxide and Fenton reagents and produced similar results. According to the data, damage of the DNA film was complete in 1 h in Fenton reagents and in 3 h in styrene oxide. In addition, the Fenton reaction induced much more severe damage than styrene oxide. The results demonstrate for the first time that the photoelectrochemical DNA sensor can detect both DNA adduct formation and DNA oxidation. It has the potential of becoming a screening tool for the rapid assessment of the genotoxicity of existing and new chemicals.

Quantitative detection of a biological affinity reaction, the biotin/avidin recognition, was achieved using our newly developed photoelectrochemical analytical system. The system is based on the operation mechanism of the well-developed dye-sensitized photoelectrochemical solar cells and comprises a ruthenium tris(2,2'-bipyridine) (Ru-bipy) derivative as the photoelectrochemical signal-generating molecule, oxalate as the sacrificial electron donor, and tin oxide nanoparticle as the semiconductor electrode material. To perform the affinity reaction, avidin was immobilized on SnO(2) electrode by passive adsorption. Biotin-linked bovine serum albumin (BSA) was labeled with an NHS-ester derivative of Ru-bipy. After binding of BSA to the surface-immobilized avidin through biotin, photoelectrochemical measurement was carried out in the presence of oxalate. Anodic photocurrent was turned on and off repeatedly by control of incidental light. The action spectrum of the photocurrent resembled the absorption spectrum of Ru-bipy, proving the photocurrent was generated from the metal complex. A linear relationship between photocurrent and BSA concentration was obtained in the range of 1-100 microg/mL. This is the first case of quantitative photoelectrochemical detection of a biological affinity interaction.

The binding interactions between ligands and receptors play a vital role in their biological functions. In this work, a label-free indirect competitive binding assay based on surface plasmon resonance (SPR) detection was developed, using fatty acid–human serum albumin (HSA) as a model system. In the assay, a fatty acid analog was immobilized on the surface of an SPR gold chip by self-assembly, and was allowed to bind with HSA in solution. Addition of a ligand under investigation into the solution set up a competition with the analog for the protein, causing a reduction in the SPR signal. Due to the asymmetric structure of the ligand binding channel, HSA exhibited remarkable selectivity for the orientation of the fatty acid analog on a gold surface. When the carboxylic acid group of the analog was exposed on the sensor surface, the amount of bound HSA was very low. However, it increased by more than 7 fold when the alkyl chain was exposed. Dilution of the surface coverage of the fatty acid analog also enhanced HSA binding by reducing steric hindrance on the surface. Using a 1 : 4 mercaptoundecanoic acid–mercaptohexanol modified surface derivatized with octylamine, competitive binding assays for octanoic acid and perfluorooctane sulfonate with HSA were conducted. Using a derived kinetic equilibrium model, the equilibrium association constant was determined to be 4.57 × 105 M−1 and 2.11 × 105 M−1, respectively, which are consistent with the values measured by other established methods. The engineered sensor chip surface can be used to study the binding interactions of other chemicals with this important serum transport protein.

Quantitation of biological affinity reactions by a newly developed chemically amplified electrochemical detection method was demonstrated with the biotin-avidin binding pair. In the method, ruthenium tris(2,2'-bipyridine)(Ru-bipy) was used as an electrochemical signal-generating tag. Its oxidation current on an indium tin oxide (ITO) electrode was amplified with a sacrificial electron donor, oxalate. Because oxalate itself produced negligible current on the electrode, the signal-to-background ratio was greatly enhanced in comparison with other chemical amplification systems. Although the Ru-bipy/oxalate redox couple has been employed previously in electrochemiluminescent and photoelectrochemical detection, its use in a catalytic amperometric detection of biological binding assays has not been reported. To implement the method in the detection of biotin-avidin recognition, avidin was immobilized on an ITO electrode, and was reacted with biotin in solution. Immobilization of avidin by passive adsorption was found to be relatively stable under the condition of the affinity reaction. In the direct assay, biotin labelled with Ru-bipy was recognized by avidin and accumulated on the electrode surface, which was then detected electrochemically in the presence of oxalate. A linear relationship between electrochemical current and biotin concentration was obtained in the range of 1-300 ng mL(-1). In the competitive assay, a mixed solution of unlabelled biotin (the analyte) of various concentrations and 100 ng mL(-1) labelled biotin was reacted with avidin on the surface. As the concentration of the unlabelled biotin increased, less labelled biotin bound to avidin, leading to a reduction in the electro-catalytical response of Ru-bipy. A detection limit of 1 ng mL(-1) biotin was obtained in the competitive assay, which is close to the sensitivity of some enzyme-labelled amperometric assays.