Journal of Analytical Methods in Chemistry

Journal of Analytical Methods in Chemistry / 2016 / Article

Research Article | Open Access

Volume 2016 |Article ID 9081375 |

Jing Li, Byung Kun Kim, Kang-Kyun Wang, Ji-Eun Im, Han Nim Choi, Dong-Hwan Kim, Seong In Cho, Won-Yong Lee, Yong-Rok Kim, "Sensing Estrogen with Electrochemical Impedance Spectroscopy", Journal of Analytical Methods in Chemistry, vol. 2016, Article ID 9081375, 6 pages, 2016.

Sensing Estrogen with Electrochemical Impedance Spectroscopy

Academic Editor: Miren Lopez de Alda
Received25 Apr 2016
Revised11 Aug 2016
Accepted04 Sep 2016
Published10 Oct 2016


This study demonstrates the application feasibility of electrochemical impedance spectroscopy (EIS) in measuring estrogen (17β-estradiol) in gas phase. The present biosensor gives a linear response () for 17β-estradiol vapor concentration from 3.7 ng/L to 3.7 × 10−4 ng/L with a limit of detection (3.7 × 10−4 ng/L). The results show that the fabricated biosensor demonstrates better detection limit of 17β-estradiol in gas phase than the previous report with GC-MS method. This estrogen biosensor has many potential applications for on-site detection of a variety of endocrine disrupting compounds (EDCs) in the gas phase.

1. Introduction

While rapid industrialization in the 20th century has introduced a significant degree of convenience to human life, the global population is being exposed to a polluted environment that includes contaminated air such as carbon monoxide, sulfur dioxide, dioxin, formaldehyde, chlorine, bisphenol, and estradiol [1]. Among the pollutants, 4-nonylphenol, bisphenol, and estradiol are classified as endocrine disrupting chemicals (EDCs) and these EDCs are known to be related to various diseases of infertility, spontaneous abortions, birth defects, endometriosis, breast cancer, and so forth [28]. Several papers have been published on the detection of EDCs in solution phase using methods such as fluorimetry, NMR spectroscopy, chromatography, enzyme linked receptor assays, and linear sweep voltammetry [917]. Recently, a few researchers studied the detection of EDCs using gas chromatography-mass spectrometry and found 4~8 ng/L of EDCs in gas phase [18]. Therefore, the development of EDC detection and measurement technique is in a great demand, particularly, at very low concentration (0.2~141 ng/L of 17β-estradiol exists in environment) in gas phase since the extremely low amount of EDCs may cause the fatal diseases to human [19]. Nevertheless, most people are not aware of the seriousness of EDCs in air. Notably, inhalation in an EDC exposed environment is an easy means of being affected by EDCs. Therefore, one of the main challenges in research is to develop a sensitive and reliable method for determination of EDCs in gas phase.

In the present study, a highly sensitive electrochemical biosensor for the detection of the EDC, 17β-estradiol, in gas phase has been developed using estrogen receptor-α immobilized on a gold electrode. 17β-estradiol in gas phase was detected by electrochemical impedance spectroscopy of at the fabricated biosensor. The present biosensor gives a linear response () for the 17β-estradiol vapor concentration from 3.7 ng/L to 3.7 × 10−4 ng/L. This study is aimed at developing a technique capable of detecting low EDC concentration (>3.7 × 10−4 ng/L) in gas phase.

2. Materials

Estrogen receptor-α (ER-α, >85.0%) was purchased from Calbiochem (San Diego, USA). Estrogen hormone (17β-estradiol, >98%), 3-mercaptopropionic acid (3-MPA, >99%), 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDC, >98%), N-hydroxysuccinimide (NHS, >98%), and bovine serum albumin (BSA, >96%) were purchased from Aldrich. A phosphate buffered saline (PBS) was purchased from Gibco (NY, USA).

For the gas phase experiment, the estrogen biosensor was prepared as previously reported [20, 21]. The fabricated estrogen biosensor was applied to the detection of 17β-estradiol in gas phase. In the experiment, a freshly pretreated gold electrode was incubated in 40 mM 3-MPA solution for 4 h to form a carboxylate terminated self-assembled monolayer (SAM) on the electrode. The electrode was then rinsed using distilled water three times to remove any nonphysically adsorbed 3-MPA molecules before it was stored in distilled water. The 3-MPA modified gold electrode was immersed in a mixture of EDC (1.0 wt.%) and NHS (1.0 wt.%) in 0.05 M PBS (pH 7.4) for 2 h to activate the terminal carboxylic groups, before it was washed using distilled water to remove any nonbonded residual EDC/NHS. Next, a 15 μL aliquot of 0.67 μM estrogen receptor-α solution was applied to the surface of the EDC-NHS/3-MPA modified gold electrode. Any nonbonded estrogen receptor-α on the EDC-NHS/3-MPA modified gold electrode was removed by rinsing using PBS. Finally, the electrode was incubated in 3.5 wt.% BSA for 4 h to block any nonspecific binding sites. Then the biosensor was dried at 25°C for 4 h and then fixed in the empty headspace of a 10 mL vial which contained only 2 mL of 17β-estradiol solution (0.01~100 mM) at the bottom of the vial in order to avoid direct contact between the biosensor and the solution. The vial was stirred at 80 rpm for 90 min (25°C, pH 7). The process was conducted in a sealed vial [2224]. The schematic diagram of the biosensor fabrication and 17β-estradiol binding is illustrated in Scheme 1.

3. Methods

Electrochemical impedance experiments were performed using an EG&G 263A potentiostat (Princeton, NJ, USA). A 15 mL electrochemical cell that accommodates a platinum wire counter electrode, the estrogen biosensor as the working electrode, and a Ag/AgCl (3 M NaCl) reference electrode was used. AC impedance spectra were recorded in 0.05 M PBS (pH 7.4) containing 5.0 mM and 5.0 mM . The impedance spectra were obtained in the frequency range from 100 MHz to 100 kHz at a bias potential of 0.2 V that was superimposed by an ac potential of 5.0 mV peak-to-peak amplitude. The impedance spectra were plotted in the form of impedance plane plots (Nyquist plots). Zsimpwin program (Princeton Applied Research, Oak Ridge, TN, USA) was used to fit theoretical impedance plots generated based on an equivalent circuit (depicted in Figure 1(c)) to those obtained experimentally. In this way, the diameter of a semicircle in the high frequency range of Nyquist plot was estimated [20, 21].

4. Results and Discussion

For the detection of 17β-estradiol in gas phase, the estrogen biosensor was exposed to 17β-estradiol vapor generated by solutions of concentration between 0.01 and 100 mM for 90 min. Based on Henry’s Law, the concentration of 17β-estradiol vapor is proportional to that of aqueous 17β-estradiol. Henry’s Law constant of 17β-estradiol is known to be 3.64 × 10−11 L·atm/mol [25]. SI unit of Henry’s Law constant was converted to unit of M/atm and the value is calculated to be 2.7 × 1010 M/atm, and the corresponding concentration of gaseous 17β-estradiol was estimated to be within the range between 0.00037 ng/L and 3.7 ng/L.

As shown in Figure 1(a), the diameter of Nyquist plots, which corresponds to the electron transfer resistance, linearly increases with 17β-estradiol concentration. This is because, with increasing 17β-estradiol concentration, a corresponding increased quantity of 17β-estradiol in the vapor was expected to bind to the immobilized estrogen receptor-α, which would in turn gradually hinder the diffusion of [Fe(CN)6]3−/[Fe(CN)6]4− to the biosensor surface. We assume that the 17β-estradiol vapor was well bonded to the ER-α on the biosensor surface. In order to normalize the data, the impedance change, , was used in the data analysis, where and represent the electron transfer resistance after and before the binding of 17β-estradiol to the estrogen biosensor, respectively.

As shown in Figure 1(b), the calibration plot was obtained by graphing the impedance change as a function of the 17β-estradiol concentration in gas phase. A linear regression equation was obtained as = 2195 (, , ). The limit of quantification of the present estrogen biosensor was determined to be 3.7 × 10−4 ng/L of 17β-estradiol.

The resistance of biosensor was measured with a CPE of electrode since the component has incorporated both the Helmholtz double layer and surface roughness or heterogeneity of the electrode. The values of were fixed at 80 ohm as shown in Table 1. CPE and were determined from the fitting program (ZsimpWin fitting software). The values of were compensated by the fitting software because the semicircle did not reach to the axis of abscissa in the low frequency region. The fitted values were presented in Table 1.

17-estradiol concentration (ng/L) (Ω) (nF) (Ω) (Ω)

3.7 × 10−4802019169823034
3.7 × 10−3801917198903219
3.7 × 10−2801979215303310
3.7 × 10−1801197256033895
3.7 802041260673068

Association constant () of the binding reaction between 17β-estradiol and estrogen receptor-α was determined using a Langmuir isotherm approach as follows [26]:where is the fractional coverage of the surface and is the concentration of 17β-estradiol vapor. Also, is expressed by the following equation between and [27]:where and represent the electron transfer resistance after and before the binding of 17β-estradiol to the estrogen biosensor, respectively. Above equations are further rearranged to

Accordingly, the ordinate intercept of a linear versus plot (as shown in Figure 2) will provide an estimate for , through which can be evaluated.

Figure 2 reveals that linearly changes with the concentration, which results in a linear equation of = 8.56 × 10−5 C + 2.63 × 10−6 (, ). From the fitting of the equation, the association constant was observed to be ca. 32.5 L/ng (8.84 × 1012 M−1). The value of is larger than the reported value of 5.2 × 108 M−1 [28]. The result implied that the binding sites on the ER-α were well maintained after the introduction of ER-α to the surface of modified electrode.

The identical biosensing measurements were carried out with corticosterone and dexamethasone that have the similar chemical structures without the bonding characteristics to 17β-estradiol [29]. The selectivity values were obtained by comparing the impedance values at the high concentrations (3.7 × 10−1 ng/L) of corticosterone and dexamethasone with those for 17β-estradiol. As shown in Figure 3, the selectivity values for corticosterone and dexamethasone are % and % due to nonspecific adsorption to the biosensor [30]. The result indicates that the fabricated estrogen biosensor in this study can be used for the selective estimation of 17β-estradiol in gas phase.

5. Conclusion

In this study, the electrochemical impedance biosensor was successfully developed by immobilizing ER-α on a Au electrode and demonstrated for the detection of the gas phase of 17β-estradiol. It is considered that such study may open the possibility of the detector development for the detection of EDCs in gas phase.

Competing Interests

The authors declare that there are no competing interests regarding the publication of this paper.

Authors’ Contributions

Jing Li, Byung Kun Kim, and Kang-Kyun Wang contributed equally to this work.


This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project no. PJ01083001),” Rural Development Administration, and the grant of the National Research Foundation of Korea (NRF) funded by the Korea government (MEST) (no. 2011-0001129), Republic of Korea.


  1. R. V. Bhatt, “Environmental influence on reproductive health,” International Journal of Gynecology & Obstetrics, vol. 70, no. 1, pp. 69–75, 2000. View at: Publisher Site | Google Scholar
  2. J. L. Liu, R. M. Wang, B. Huang, C. Lin, Y. Wang, and X. J. Pan, “Distribution and bioaccumulation of steroidal and phenolic endocrine disrupting chemicals in wild fish species from Dianchi Lake, China,” Environmental Pollution, vol. 159, no. 10, pp. 2815–2822, 2011. View at: Publisher Site | Google Scholar
  3. V. Granek and J. Rishpon, “Detecting endocrine-disrupting compounds by fast impedance measurements,” Environmental Science and Technology, vol. 36, no. 7, pp. 1574–1578, 2002. View at: Publisher Site | Google Scholar
  4. J. G. Liehr, “Is estradiol a genotoxic mutagenic carcinogen?” Endocrine Reviews, vol. 21, no. 1, pp. 40–54, 2000. View at: Publisher Site | Google Scholar
  5. X. Wang, L. Yang, X. Jin, and L. Zhang, “Electrochemical determination of estrogenic compound bisphenol F in food packaging using carboxyl functionalized multi-walled carbon nanotubes modified glassy carbon electrode,” Food Chemistry, vol. 157, pp. 464–469, 2014. View at: Publisher Site | Google Scholar
  6. K. V. Ragavan, N. K. Rastogi, and M. S. Thakur, “Sensors and biosensors for analysis of bisphenol-A,” TrAC—Trends in Analytical Chemistry, vol. 52, pp. 248–260, 2013. View at: Publisher Site | Google Scholar
  7. P. Nicolopoulou-Stamati and M. A. Pitsos, “The impact of endocrine disrupters on the female reproductive system,” Human Reproduction Update, vol. 7, no. 3, pp. 323–330, 2001. View at: Publisher Site | Google Scholar
  8. H. Hamid and C. Eskicioglu, “Fate of estrogenic hormones in wastewater and sludge treatment: a review of properties and analytical detection techniques in sludge matrix,” Water Research, vol. 46, no. 18, pp. 5813–5833, 2012. View at: Publisher Site | Google Scholar
  9. J. Fan, H. Q. Guo, G. G. Liu, and P. G. Peng, “Simple and sensitive fluorimetric method for determination of environmental hormone bisphenol A based on its inhibitory effect on the redox reaction between peroxyl radical and rhodamine 6G,” Analytica Chimica Acta, vol. 585, no. 1, pp. 134–138, 2007. View at: Publisher Site | Google Scholar
  10. C. Jung, J. Park, K. H. Lim et al., “Adsorption of selected endocrine disrupting compounds and pharmaceuticals on activated biochars,” Journal of Hazardous Materials, vol. 263, pp. 702–710, 2013. View at: Publisher Site | Google Scholar
  11. A. Gómez-Hens and M. P. Aguilar-Caballos, “Social and economic interest in the control of phthalic acid esters,” TrAC—Trends in Analytical Chemistry, vol. 22, no. 11, pp. 847–857, 2003. View at: Publisher Site | Google Scholar
  12. H. Matsumoto, S. Adachi, and Y. Suzuki, “Bisphenol A in ambient air particulates responsible for the proliferation of MCF-7 human breast cancer cells and its concentration changes over 6 months,” Archives of Environmental Contamination and Toxicology, vol. 48, no. 4, pp. 459–466, 2005. View at: Publisher Site | Google Scholar
  13. J. Lu, J. Wu, P. J. Stoffella, and P. C. Wilson, “Isotope dilution-gas chromatography/mass spectrometry method for the analysis of alkylphenols, bisphenol A, and estrogens in food crops,” Journal of Chromatography A, vol. 1258, pp. 128–135, 2012. View at: Publisher Site | Google Scholar
  14. M. Hansen, K. A. Krogh, B. Halling-Sørensen, and E. Björklund, “Determination of ten steroid hormones in animal waste manure and agricultural soil using inverse and integrated clean-up pressurized liquid extraction and gas chromatography-tandem mass spectrometry,” Analytical Methods, vol. 3, no. 5, pp. 1087–1095, 2011. View at: Publisher Site | Google Scholar
  15. C. Pieper and W. Rotard, “Investigation on the removal of natural and synthetic estrogens using biofilms in continuous flow biofilm reactors and batch experiments analysed by gas chromatography/mass spectrometry,” Water Research, vol. 45, no. 3, pp. 1105–1114, 2011. View at: Publisher Site | Google Scholar
  16. D. Habauzit, A. Boudot, G. Kerdivel, G. Flouriot, and F. Pakdel, “Development and validation of a test for environmental estrogens: checking xeno-estrogen activity by CXCL12 secretion in Breast Cancer Cell Lines (CXCL-test),” Environmental Toxicology, vol. 25, no. 5, pp. 495–503, 2010. View at: Publisher Site | Google Scholar
  17. J. H. Li, D. Z. Kuang, Y. L. Feng, F. X. Zhang, and M. Q. Liu, “Voltammetric determination of bisphenol A in food package by a glassy carbon electrode modified with carboxylated multi-walled carbon nanotubes,” Microchimica Acta, vol. 172, no. 3-4, pp. 379–386, 2011. View at: Publisher Site | Google Scholar
  18. R. A. Pérez, B. Albero, J. L. Tadeo, E. Molero, and C. Sánchez-Brunete, “Analysis of steroid hormones in water using palmitate-coated magnetite nanoparticles solid-phase extraction and gas chromatography-tandem mass spectrometry,” Chromatographia, vol. 77, no. 11-12, pp. 837–843, 2014. View at: Publisher Site | Google Scholar
  19. R. L. Gomes and J. N. Lester, “Endocrine disrupters in drinking water and water reuse,” in Endocrine Disruptors in Wastewater and Sludge Treatment Processes, J. W. Birkett and J. N. Lester, Eds., pp. 177–218, CRC Press, London, UK, 2002. View at: Google Scholar
  20. J.-E. Im, J.-A. Han, B. K. Kim et al., “Electrochemical detection of estrogen hormone by immobilized estrogen receptor on Au electrode,” Surface and Coatings Technology, vol. 205, supplement 1, pp. S275–S278, 2010. View at: Publisher Site | Google Scholar
  21. B. K. Kim, J. Li, J.-E. Im et al., “Impedometric estrogen biosensor based on estrogen receptor alpha-immobilized gold electrode,” Journal of Electroanalytical Chemistry, vol. 671, pp. 106–111, 2012. View at: Publisher Site | Google Scholar
  22. I. Goubaidoulline, G. Vidrich, and D. Johannsmann, “Organic vapor sensing with ionic liquids entrapped in alumina nanopores on quartz crystal resonators,” Analytical Chemistry, vol. 77, no. 2, pp. 615–619, 2005. View at: Publisher Site | Google Scholar
  23. S. Tao, L. Xu, and J. C. Fanguy, “Optical fiber ammonia sensing probes using reagent immobilized porous silica coating as transducers,” Sensors and Actuators B: Chemical, vol. 115, no. 1, pp. 158–163, 2006. View at: Publisher Site | Google Scholar
  24. M. Hämmerle, K. Hilgert, M. A. Horn, and R. Moos, “Analysis of volatile alcohols in apple juices by an electrochemical biosensor measuring in the headspace above the liquid,” Sensors and Actuators B: Chemical, vol. 158, no. 1, pp. 313–318, 2011. View at: Publisher Site | Google Scholar
  25. M. Kuster, M. José López De Alda, and D. Barceló, “Analysis and distribution of estrogens and progestogens in sewage sludge, soils and sediments,” TrAC—Trends in Analytical Chemistry, vol. 23, no. 10-11, pp. 790–798, 2004. View at: Publisher Site | Google Scholar
  26. Y.-K. Lyu, K.-R. Lim, B. Y. Lee, K. S. Kim, and W.-Y. Lee, “Microgravimetric lectin biosensor based on signal amplification using carbohydrate-stabilized gold nanoparticles,” Chemical Communications, no. 39, pp. 4771–4773, 2008. View at: Publisher Site | Google Scholar
  27. P. Li, B. Ge, L. M. L. Ou, Z. Yao, and H.-Z. Yu, “DNA-redox cation interaction improves the sensitivity of an electrochemical immunosensor for protein detection,” Sensors, vol. 15, no. 8, pp. 20543–20556, 2015. View at: Publisher Site | Google Scholar
  28. K. Hattori, T. Takeuchi, M. Ogata et al., “Detection of environmental chemicals by SPR assay using branched cyclodextrin as sensor ligand,” Journal of Inclusion Phenomena and Macrocyclic Chemistry, vol. 57, no. 1–4, pp. 339–342, 2007. View at: Publisher Site | Google Scholar
  29. R. L. Rich, L. R. Hoth, K. F. Geoghegan et al., “Kinetic analysis of estrogen receptor/ligand interactions,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 13, pp. 8562–8567, 2002. View at: Publisher Site | Google Scholar
  30. S. K. Arya, G. Chornokur, M. Venugopal, and S. Bhansali, “Dithiobis(succinimidyl propionate) modified gold microarray electrode based electrochemical immunosensor for ultrasensitive detection of cortisol,” Biosensors and Bioelectronics, vol. 25, no. 10, pp. 2296–2301, 2010. View at: Publisher Site | Google Scholar

Copyright © 2016 Jing Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

More related articles

 PDF Download Citation Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly as possible. We will be providing unlimited waivers of publication charges for accepted research articles as well as case reports and case series related to COVID-19. Review articles are excluded from this waiver policy. Sign up here as a reviewer to help fast-track new submissions.