Ultrasensitive Electrochemical Biosensor for Rapid Screening of Chemicals with Estrogenic Effect

Abstract

Estrogenic chemicals are widely distributed and structurally diverse. They primarily disrupt estrogen-related metabolism in animals or humans by mimicking the agonistic receptor effects of natural estrogens, thereby influencing the transcription of estrogen receptors to regulate their quantity and sensitivity. This disruption of estrogen-related metabolism can lead to estrogen-related effects, posing risks to biological health, emphasizing the urgent need for simple and effective methods to screen compounds with estrogenic effects. Herein, a new electrochemical biological effect biosensor based on human estrogen receptor α (hERα) is developed, which uses hERα as the biorecognition element and employs the electroactive horseradish peroxidase (HRP) labeled 17β-estradiol (E2) multifunctional conjugate HRP-E2 as the signal-boosting element and ligand competition agent. Based on the specific ligand-receptor interaction principle between the target and nuclear receptor, by allowing the test compound to compete with HRP-E2 conjugate for binding to hERα and testing the electrocatalytic signal of the conjugate that fails to bind to the hERα estrogen receptor, rapid screening and quantitative detection of chemical substances with estrogenic effect have been achieved. The biosensor shows a wide linear range of 40 pM to 40 nM with a detection limit of 17 pM (S/N = 3) for E2, and the detection limit is 2 orders of magnitude better than that of the previously reported sensors. The biosensor based on ligand-receptor binding can not only quantitatively analyze the typical estrogen E2, but also evaluate the relative estrogen effect strength of other estrogen compounds, which has good stability and selectivity. This electrochemical sensing platform displays its promising potential for rapid screening and quantitative detection of chemicals with estrogenic effects.

Keywords: electrochemical biosensor; endocrine disrupting chemicals (EDCs); estrogen; estrogenic activity evaluation; human estrogen receptor α.

Figure 1

Schematic diagram of the sample preparation (top row) and biosensing detection (bottom row). (I) Incubate for 2 h to allow full binding of hER-α with Ni-NTA-agarose resin; (II) Add E2-HRP and the compound to be tested then incubate for 1 h; (III) Centrifuge at 3000 rpm for 3 min then take the supernatant and modify it on the GCE electrode; (IV) Electrochemical biosensing signals boosted by electrocatalytic reaction between E2-HRP and TMB substrate solution.

Figure 2

(a) UV-Vis absorption spectra of TMB substrate solution (containing 0.2 mM TMB and 0.04% (v:v) H₂O₂) before and after the catalytic reaction of E2-HRP (1 mg/mL) for TMB. (b) The electrochemical cyclic voltammograms of the TMB substrate solution after the addition of various concentrations of E2-HRP.

Figure 3

(a) The UV absorption spectra of the TMB substrate solution (containing 0.2 mM TMB and 0.04% (v:v) H₂O₂) after reaction with supernatants incubated in the presence (red) or absence (black) of 1mg/mL hERα. (b) The DPV curves of the different supernatants (incubated with or without hERα) modified electrodes for the TMB substrate solution.

Figure 4

(a) The electrochemical DPV curves of the biosensor for different concentrations of E2. (b) The linear fitting curves of the response current change (%) vs. the logarithmic concentrations of E2.

Figure 5

(a) Structures of E2, E1, E3, BPA, Te and T3. (b) The response signal comparison chart of E2, E1, E3, BPA, Te, and T3 at concentrations of 1nM and 10nM.