Prêt-à-porter nanoYESα and nanoYESβ bioluminescent cell biosensors for ultrarapid and sensitive screening of endocrine-disrupting chemicals

  • Antonia Lopreside
  • Maria Maddalena Calabretta
  • Laura Montali
  • Maura Ferri
  • Annalisa Tassoni
  • Bruce R. Branchini
  • Tara Southworth
  • Marcello D’Elia
  • Aldo Roda
  • Elisa MicheliniEmail author
Research Paper
Part of the following topical collections:
  1. Young Investigators in (Bio-)Analytical Chemistry


Cell-based assays utilizing reporter gene technology have been widely exploited for biosensing, as they provide useful information about the bioavailability and cell toxicity of target analytes. The long assay time due to gene transcription and translation is one of the main drawbacks of cell biosensors. We report the development of two yeast biosensors stably expressing human estrogen receptors α and β and employing NanoLuc as the reporter protein to upgrade the widely used yeast estrogen screening (YES) assays. A viability control strain was also developed based on a chimeric green-emitting luciferase, PLG2, expressed for the first time in Saccharomycescerevisiae. Thanks to their brightness, NanoLuc and PLG2 provided excellent sensitivity, enabling the implementation of these biosensors into low-cost smartphone-based devices. The developed biosensors had a rapid (1 h) response and reported on (anti)estrogenic activity via human estrogen receptors α and β as well as general sample toxicity. Under optimized conditions, we obtained LODs of 7.1 ± 0.4 nM and 0.38 ± 0.08 nM for E2 with nanoYESα and nanoYESβ, respectively. As a proof of concept, we analyzed real samples from plants showing significant estrogenic activity or known to contain significant amounts of phytoestrogens.

Graphical abstract


Bioluminescence Biosensor Luciferase Smartphone Estrogen receptor 



We wish to thank Dr. Alessandro Filippini and Dr. Dario Zanichelli (Phenbiox Srl, Bologna, Italy) for providing soybean seed extracts, and Dr. Matteo Lamborghini (Alfavita Srl, Ravenna, Italy) for providing alfalfa plant extracts.

Funding information

This research was sponsored in part by PRIN 2015 “Multifunctional nanotools for advanced cancer diagnostics” (Prot. 2015TWP83Z), PRIN 2015 “Securing and ensuring sustainable use of agriculture waste, co-and by-products: an integrated analytical approach combining mass spectrometry with health effect-based biosensing” (Prot. 2015FFY97L), the NATO Science for Peace and Security Programme under Grant No. 985042, Air Force Office of Scientific Research (FA9950-18-1-0017) and National Science Foundation (MCB-1410390).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_1805_MOESM1_ESM.pdf (381 kb)
ESM 1 (PDF 381 kb)


  1. 1.
    Browne P, Noyes PD, Casey WM, Dix DJ. Application of adverse outcome pathways to U.S. EPA’s Endocrine Disruptor Screening Program. Environ Health Perspect. 2017;125:096001.CrossRefGoogle Scholar
  2. 2.
    De Coster S, van Larebeke N. Endocrine-disrupting chemicals: associated disorders and mechanisms of action. J Environ Public Health. 2012;2012:713696.CrossRefGoogle Scholar
  3. 3.
    Hotchkiss AK, Rider CV, Blystone CR, Wilson VS, Hartig PC, Ankley GT, et al. Fifteen years after “Wingspread”--environmental endocrine disrupters and human and wildlife health: where we are today and where we need to go. Toxicol Sci. 2008;105:235–59.CrossRefGoogle Scholar
  4. 4.
    Trasande L, Zoeller RT, Hass U, Kortenkamp A, Grandjean P, Myers JP, et al. Estimating burden and disease costs of exposure to endocrine-disrupting chemicals in the European Union. J Clin Endocrinol Metab. 2015;100:1245–55.CrossRefGoogle Scholar
  5. 5.
    Fang TY, Praveena SM, deBurbure C, Aris AZ, Ismail SNS, Rasdi I. Analytical techniques for steroid estrogens in water samples - a review. Chemosphere. 2016;165:358–68.CrossRefGoogle Scholar
  6. 6.
    Sadik OA, Wanekaya AK, Andreescu S. Advances in analytical technologies for environmental protection and public safety. J Environ Monit. 2004;6:513–22.CrossRefGoogle Scholar
  7. 7.
    Turner AP. Biosensors: sense and sensibility. Chem Soc Rev. 2013;42:3184–96.CrossRefGoogle Scholar
  8. 8.
    Elad T, Belkin S. Reporter gene assays in ecotoxicology. Adv Biochem Eng Biotechnol. 2017;157:135–57.Google Scholar
  9. 9.
    Charrier T, Chapeau C, Bendria L, Picart P, Daniel P, Thouand G. A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part II: technical development and proof of concept of the biosensor. Anal Bioanal Chem. 2011;400:1061–70.CrossRefGoogle Scholar
  10. 10.
    Roda A, Cevenini L, Michelini E, Branchini BR. A portable bioluminescence engineered cell-based biosensor for on-site applications. Biosens Bioelectron. 2011;26:3647–53.CrossRefGoogle Scholar
  11. 11.
    Ben-Yoav H, Melamed S, Freeman A, Shacham-Diamand Y, Belkin S. Whole-cell biochips for bio-sensing: integration of live cells and inanimate surfaces. Crit Rev Biotechnol. 2011;31:337–53.CrossRefGoogle Scholar
  12. 12.
    Michelini E, Calabretta MM, Cevenini L, Lopreside A, Southworth T, Fontaine DM, et al. Smartphone-based multicolor bioluminescent 3D spheroid biosensors for monitoring inflammatory activity. Biosens Bioelectron. 2019;123:269–77.CrossRefGoogle Scholar
  13. 13.
    Elad T, Belkin S. Whole-cell biochips for online water monitoring. Bioeng Bugs. 2012;3:124–8.Google Scholar
  14. 14.
    Zhang D, He Y, Wang Y, Wang H, Wu L, Aries E, et al. Whole-cell bacterial bioreporter for actively searching and sensing of alkanes and oil spills. Microb Biotechnol. 2012;5:87–97.CrossRefGoogle Scholar
  15. 15.
    Jouanneau S, Durand MJ, Thouand G. Online detection of metals in environmental samples: comparing two concepts of bioluminescent bacterial biosensors. Environ Sci Technol. 2012;46:11979–87.CrossRefGoogle Scholar
  16. 16.
    Cevenini L, Michelini E, D’Elia M, Guardigli M, Roda A. Dual-color bioluminescent bioreporter for forensic analysis: evidence of androgenic and anti-androgenic activity of illicit drugs. Anal Bioanal Chem. 2013;405:1035–45.CrossRefGoogle Scholar
  17. 17.
    LaLone C, Villeneuve DL, Doering JA, Blackwell BR, Transue TR, Simmons CW, et al. Evidence for cross species extrapolation of mammalian-based high-throughput screening assay results. Environ Sci Technol. 2018. in press.
  18. 18.
    Michelini E, Cevenini L, Mezzanotte L, Coppa A, Roda A. Cell-based assays: fuelling drug discovery. Anal Bioanal Chem. 2010;398:227–38.CrossRefGoogle Scholar
  19. 19.
    Jarque S, Bittner M, Blaha L, Hilscherova K. Yeast biosensors for detection of environmental pollutants: current state and limitations. Trends Biotechnol. 2016;34:408–19.CrossRefGoogle Scholar
  20. 20.
    Cevenini L, Lopreside A, Calabretta MM, D’Elia M, Simoni P, Michelini E, et al. A novel bioluminescent NanoLuc yeast-estrogen screen biosensor (nanoYES) with a compact wireless camera for effect-based detection of endocrine-disrupting chemicals. Anal Bioanal Chem. 2018;410:1237–46.CrossRefGoogle Scholar
  21. 21.
    Masser AE, Kandasamy G, Kaimal JM, Andréasson C. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae. Yeast. 2016;33:191–200.CrossRefGoogle Scholar
  22. 22.
    Branchini BR, Southworth TL, Fontaine DM, Kohrt D, Talukder M, Michelini E, et al. An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications. Anal Biochem. 2015;484:148–53.CrossRefGoogle Scholar
  23. 23.
    Schena M, Picard D, Yamamoto KR. Vectors for constitutive and inducible gene expression in yeast. Methods Enzymol. 1991;194:389–98.CrossRefGoogle Scholar
  24. 24.
    Baudin-Bailleu A, Guillemet E, Cullin C, Lacroute F. Construction of a yeast strain deleted for the TRP1 promoter and coding region that enhances the efficiency of the polymerase chain reaction disruption method. Yeast. 1996;30:353–6.Google Scholar
  25. 25.
    Sambrook EF, Fristch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989.Google Scholar
  26. 26.
    Sansanelli S, Zanichelli D, Filippini A, Ferri M, Tassoni A. Production of free and glycosylated isoflavones in in vitro soybean (Glycine max L.) hypocotyl cell suspensions and comparison with industrial seed extracts. Plant Cell Tissue Organ Cult. 2014;119:301–11.CrossRefGoogle Scholar
  27. 27.
    Leclerc GM, Boockfor FR, Faught WJ, Frawley LS. Development of a destabilized firefly luciferase enzyme for measurement of gene expression. Biotechniques. 2000;29(3):590–1 594-6, 598 passim.CrossRefGoogle Scholar
  28. 28.
    Routledge EJ, Sumpter JP. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ Toxicol Chem. 1996;15:241–8.CrossRefGoogle Scholar
  29. 29.
    Rajapakse N, Ong D, Kortenkamp A. Defining the impact of weakly estrogenic chemicals on the action of steroidal estrogens. Toxicol Sci. 2001;60:296–304.CrossRefGoogle Scholar
  30. 30.
    Balsiger HA, de la Torre R, Lee WY, Cox MB. A four-hour yeast bioassay for the direct measure of estrogenic activity in wastewater without sample extraction, concentration, or sterilization. Sci Total Environ. 2010;408:1422–9.CrossRefGoogle Scholar
  31. 31.
    Bovee TF, Helsdingen RJ, Rietjens IM, Keijer J, Hoogenboom RL. Rapid yeast estrogen bioassays stably expressing human estrogen receptors alpha and beta, and green fluorescent protein: a comparison of different compounds with both receptor types. J Steroid Biochem Mol Biol. 2004;91:99–109.CrossRefGoogle Scholar
  32. 32.
    Sanseverino J, Gupta RK, Layton AC, Patterson SS, Ripp SA, Saidak L, et al. Use of Saccharomyces cerevisiae BLYES expressing bacterial bioluminescence for rapid, sensitive detection of estrogenic compounds. Appl Environ Microbiol. 2005;71:4455–60.CrossRefGoogle Scholar
  33. 33.
    Bergamasco AM, Eldridge M, Sanseverino J, Sodré FF, Montagner CC, Pescara IC, et al. Bioluminescent yeast estrogen assay (BLYES) as a sensitive tool to monitor surface and drinking water for estrogenicity. J Environ Monit. 2011;13(11):3288–93.CrossRefGoogle Scholar
  34. 34.
    Leskinen P, Michelini E, Picard D, Karp M, Virta MP. Bioluminescent yeast assays for detecting estrogenic and androgenic activity in different matrices. Chemosphere. 2005;61:259–66.CrossRefGoogle Scholar
  35. 35.
    Escande A, Pillon A, Servant N, Cravedi JP, Larrea F, Muhn P, et al. Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta. Biochem Pharmacol. 2006;71:1459–69.CrossRefGoogle Scholar
  36. 36.
    Lee GS, Choi KC, Kim HJ, Jeung EB. Effect of genistein as a selective estrogen receptor beta agonist on the expression of calbindin-D9k in the uterus of immature rats. Toxicol Sci. 2004;82:451–7.CrossRefGoogle Scholar
  37. 37.
    Hettwer K, Jähne M, Frost K, Giersberg M, Kunze G, Trimborn M, et al. Validation of Arxula yeast estrogen screen assay for detection of estrogenic activity in water samples: results of an international interlaboratory study. Sci Total Environ. 2018;621:612–25.CrossRefGoogle Scholar
  38. 38.
    European Commission. Common implementation strategy for the Water Framework Directive (2000/60/EC) guidance on surface water chemical monitoring under the Water Framework Directive. Guidance Document No. 19; 2009–025.Google Scholar
  39. 39.
    Cevenini L, Calabretta MM, Lopreside A, Tarantino G, Tassoni A, Ferri M, et al. Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity. Anal Bioanal Chem. 2016;408:8859–68.CrossRefGoogle Scholar
  40. 40.
    Kim H, Jung Y, Doh IJ, Lozano-Mahecha RA, Applegate B, Bae E. Smartphone-based low light detection for bioluminescence application. Sci Rep. 2017;7:40203.CrossRefGoogle Scholar
  41. 41.
    Cevenini L, Calabretta MM, Tarantino G, Michelini E, Roda A. Smartphone-interfaced 3D printed toxicity biosensor integrating bioluminescent “sentinel cells”. Sensors Actuators B Chem. 2016;225:249–5.CrossRefGoogle Scholar
  42. 42.
    Boué SM, Wiese TE, Nehls S, Burow ME, Elliott S, Carter-Wientjes CH, et al. Evaluation of the estrogenic effects of legume extracts containing phytoestrogens. J Agric Food Chem. 2003;51(8):2193–9.CrossRefGoogle Scholar
  43. 43.
    Ferri M, Graen-Heedfeld J, Bretz K, Guillon F, Michelini E, Calabretta MM, et al. Peptide fractions obtained from rice by-products by means of an environment-friendly process show in vitro health-related bioactivities. PLoS One. 2017;12:e0170954.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Antonia Lopreside
    • 1
  • Maria Maddalena Calabretta
    • 1
  • Laura Montali
    • 1
  • Maura Ferri
    • 2
    • 3
  • Annalisa Tassoni
    • 2
  • Bruce R. Branchini
    • 4
  • Tara Southworth
    • 4
  • Marcello D’Elia
    • 5
  • Aldo Roda
    • 1
    • 6
  • Elisa Michelini
    • 1
    • 6
    • 7
    Email author
  1. 1.Department of Chemistry “G. Ciamician”University of BolognaBolognaItaly
  2. 2.Department of Biological Geological and Environmental Sciences (BIGeA)University of BolognaBolognaItaly
  3. 3.Department of Civil, Chemical, Environmental and Materials EngineeringUniversity of BolognaBolognaItaly
  4. 4.Department of ChemistryConnecticut CollegeNew LondonUSA
  5. 5.Gabinetto Regionale di Polizia Scientifica per l’Emilia-RomagnaBolognaItaly
  6. 6.INBB, Istituto Nazionale di Biostrutture e BiosistemiRomeItaly
  7. 7.Health Sciences and Technologies-Interdepartmental Center for Industrial Research (HST-ICIR)University of BolognaOzzano dell’EmiliaItaly

Personalised recommendations