Label-Free Biosensor Detection of Endocrine Disrupting Compounds Using Engineered Estrogen Receptors

Endocrine Disrupting Compounds (EDCs) are chemical substances shown to interfere with endogenous hormones affecting the endocrine, immune and nervous systems of mammals. EDCs are the causative agents of diseases including reproductive disorders and cancers. This highlights the urgency to develop fast and sensitive methods to detect EDCs, which are detrimental even at very low concentrations. In this work, we propose a label-free surface plasmon resonance (SPR) biosensor method to detect specific EDCs (17 β-estradiol (E2), ethinyl-estradiol, 4-nonylphenol, tamoxifen) through their binding to estrogen receptor alpha (ERα). We show that the use of rationally designed ERα (as bio-recognition element) in combination with conformation-sensitive peptides (as amplification agent, resulting in increased responses) enables the detection of low parts per billion (ppb) levels of E2. As a proof of concept, this bioassay was used to detect E2 in (spiked) real water samples from fish farms, rivers and the sea at low ppb levels after concentration by solid phase extraction. In addition, the present SPR assay that combines a conformation-sensitive peptide with an array of ERα mutants is very promising for the assessment of the risk of potential estrogenic activity for chemical substances.

[1]  A. Cooney,et al.  The varied roles of nuclear receptors during vertebrate embryonic development , 2003, Nuclear receptor signaling.

[2]  F. Labrie,et al.  A sensitive, simple and robust LC–MS/MS method for the simultaneous quantification of seven androgen- and estrogen-related steroids in postmenopausal serum , 2014, The Journal of Steroid Biochemistry and Molecular Biology.

[3]  M Carlquist,et al.  Structure of the ligand‐binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist , 1999, The EMBO journal.

[4]  L. Varani,et al.  Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry , 2013, Analytical and Bioanalytical Chemistry.

[5]  G. Valesini,et al.  Autoantibodies to estrogen receptor α interfere with T lymphocyte homeostasis and are associated with disease activity in systemic lupus erythematosus. , 2012, Arthritis and rheumatism.

[6]  Gene regulation by steroid hormones , 1989, Cell.

[7]  D. Marino,et al.  Screening Concentration of E1, E2 and EE2 in Sewage Effluents and Surface Waters of the “Pampas” Region and the “Río de la Plata” Estuary (Argentina) , 2014, Bulletin of Environmental Contamination and Toxicology.

[8]  G. Martin,et al.  Estrogen receptor-associated proteins: possible mediators of hormone-induced transcription. , 1994, Science.

[9]  Luigi Calzolai,et al.  Rational Modification of Estrogen Receptor by Combination of Computational and Experimental Analysis , 2014, PloS one.

[10]  Julie E. Goodman,et al.  A critique of the European Commission Document, “State of the Art Assessment of Endocrine Disrupters” , 2012, Critical reviews in toxicology.

[11]  Amina Antonacci,et al.  Analytical tools monitoring endocrine disrupting chemicals , 2016 .

[12]  Richard David Evans,et al.  State of the art assessment of endocrine disruptors: Final Report , 2011 .

[13]  Benita S Katzenellenbogen,et al.  Estrogen receptor transcription and transactivation: Estrogen receptor alpha and estrogen receptor beta - regulation by selective estrogen receptor modulators and importance in breast cancer , 2000, Breast Cancer Research.

[14]  Dan Wu,et al.  Metal ions-based immunosensor for simultaneous determination of estradiol and diethylstilbestrol. , 2014, Biosensors & bioelectronics.

[15]  P. Andreasen,et al.  Novel modes of oestrogen receptor agonism and antagonism by hydroxylated and chlorinated biphenyls, revealed by conformation-specific peptide recognition patterns , 2008, Molecular and Cellular Endocrinology.

[16]  Jeho Park,et al.  Surface Plasmon Resonance: A Versatile Technique for Biosensor Applications , 2015, Sensors.

[17]  P. Dittrich,et al.  Automated and portable solid phase extraction platform for immuno-detection of 17β-estradiol in water. , 2015, Journal of chromatography. A.

[18]  A. Z. Aris,et al.  Endocrine disrupting compounds (EDCs) in environmental matrices: Review of analytical strategies for pharmaceuticals, estrogenic hormones, and alkylphenol compounds , 2016 .

[19]  P. Yáñez‐Sedeño,et al.  Electrochemical immunosensor for rapid and sensitive determination of estradiol. , 2012, Analytica chimica acta.

[20]  M. Febbraio,et al.  Myeloid-specific estrogen receptor α deficiency impairs metabolic homeostasis and accelerates atherosclerotic lesion development , 2011, Proceedings of the National Academy of Sciences.

[21]  B. Katzenellenbogen,et al.  Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein. , 1996, Molecular endocrinology.

[22]  G Gauglitz,et al.  Comparison of two fluorescence immunoassay methods for the detection of endocrine disrupting chemicals in water. , 2002, Biomolecular engineering.

[23]  Jennifer A. Fronczak,et al.  Analysis of ligand-dependent recruitment of coactivator peptides to estrogen receptor using fluorescence polarization. , 2005, Molecular endocrinology.

[24]  James X. Sun,et al.  Emergence of Constitutively Active Estrogen Receptor-α Mutations in Pretreated Advanced Estrogen Receptor–Positive Breast Cancer , 2014, Clinical Cancer Research.

[25]  Makoto Usami,et al.  Estrogen receptor binding assay of chemicals with a surface plasmon resonance biosensor , 2002, The Journal of Steroid Biochemistry and Molecular Biology.

[26]  L. Giudice,et al.  Endocrine-disrupting chemicals: an Endocrine Society scientific statement. , 2009, Endocrine reviews.

[27]  R. Cifelli,et al.  Analytical methods for the endocrine disruptor compounds determination in environmental water samples. , 2016, Journal of chromatography. A.

[28]  J. Scott,et al.  Searching for peptide ligands with an epitope library. , 1990, Science.

[29]  D. Fowlkes,et al.  Peptide antagonists of the human estrogen receptor. , 1999, Science.

[30]  D. O'Shannessy,et al.  Immobilization chemistries suitable for use in the BIAcore surface plasmon resonance detector. , 1992, Analytical biochemistry.

[31]  David A. Agard,et al.  Structural characterization of a subtype-selective ligand reveals a novel mode of estrogen receptor antagonism , 2002, Nature Structural Biology.

[32]  R. Waring,et al.  Endocrine disrupters: A human risk? , 2005, Molecular and Cellular Endocrinology.

[33]  Shimshon Belkin,et al.  Mixtures of Chemical Pollutants at European Legislation Safety Concentrations: How Safe Are They? , 2014, Toxicological sciences : an official journal of the Society of Toxicology.

[34]  M. Poutanen,et al.  Increased exposure to estrogens disturbs maturation, steroidogenesis, and cholesterol homeostasis via estrogen receptor alpha in adult mouse Leydig cells. , 2009, Endocrinology.

[35]  R. Schneider,et al.  Field study using two immunoassays for the determination of estradiol and ethinylestradiol in the aquatic environment. , 2006, Water research.

[36]  Willem Haasnoot,et al.  Biosensor immunoassays for the detection of bisphenol A , 2005 .

[37]  P. Andreasen,et al.  A novel pesticide‐induced conformational state of the oestrogen receptor ligand‐binding domain, detected by conformation‐specific peptide binding , 2005, FEBS letters.

[38]  Raj Kumar,et al.  The structure of the nuclear hormone receptors , 1999, Steroids.

[39]  Rebecca L Rich,et al.  Kinetic analysis of estrogen receptor/ligand interactions , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Lester,et al.  Endocrine Disrupters in Drinking Water and Water Reuse , 2003 .

[41]  Xi Chen,et al.  Design and Performance of a Portable and Multichannel SPR Device , 2017, Sensors.

[42]  J. Gustafsson,et al.  Role of estrogen receptor beta in estrogen action. , 2001, Annual review of physiology.

[43]  H Grøn,et al.  Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[44]  Yu Gao,et al.  Assessment of estrogenic activity of perfluoroalkyl acids based on ligand-induced conformation state of human estrogen receptor. , 2013, Environmental science & technology.

[45]  Tao Dong,et al.  Ultrasensitive opto-microfluidic immunosensor integrating gold nanoparticle–enhanced chemiluminescence and highly stable organic photodetector , 2014, Journal of biomedical optics.

[46]  P. Neven,et al.  Need for estradiol assays with a lower functional sensitivity in clinical studies examining postmenopausal women treated with aromatase inhibitors. , 2013, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.