Figure 1. Overview of the electrochemical sandwich assay. (a) Monobodies assembled on a gold electrode surface capture EDCs bound to estrogen receptor α (ERα) that is surface expressed on E. coli . The binding of the large bacterial cells is easily detected by impedance spectroscopy. (b) The device is constructed on disposable electrodes and requires a 10 μL sample volume. Scanning electron microscopy images are shown for the monobody-modified gold electrode surface treated with lyophilized E. coli (c) in the absence of estradiol and (d) in the presence of 10 μM estradiol. Scale bars represent 1 μm.

The approach described herein is based on a novel electrochemical sandwich assay ( Figure 1 a) and involves the use of lyophilizedto cause changes in the surface impedance upon binding. Several unique aspects of this strategy enable the detection of a range of estrogenic compounds at exceptionally low concentrations. Thesurfaces are engineered to display the ERα capture agent, which facilitates detection of any compounds that associate with its binding pocket. (23) The use of lyophilizedlimits their viability and increases storage life. The second component of the sandwich assay is an electrochemical working electrode modified with a previously reported protein that binds to ERα only when a ligand is present. (24, 25) This protein is attached through the interactions of a cysteine thiol with a disposable gold electrode surface ( Figure 1 b). The specificity of the monobody is observable by scanning electron microscopy of the working electrode surfaces. In the presence of estradiol (E2),was observed on the surface, while in the absence of E2, nobound the surface ( Figure 1 c,d).

The current standards for EDC detection are cell-based assays (originally the E-SCREEN assay (14) and, more recently, transactivation assays (15, 16) and yeast-based assays (17, 18) ) and radioactive (19) and fluorescent competition assays. (20, 21) The cell-based transactivation involves the transcription of a reporter gene, such as a luciferase gene, following the addition of the compound in question. While effective, this analytical method is problematic for rapid, point-of-care application, as it can require multiple days of cell culture, specialized equipment, and trained laboratory personnel. Similar problems arise with fluorescent polarization assays, in which fluorescently labeled 17β-estradiol is displaced from specific antibodies by estrogenic compounds. This method requires several conjugation reactions and optimization steps, and a specialized fluorometer is necessary for measurement. As alternatives, efforts have been made to develop rapid EDC detectors, including both fluorescent and electrochemical sensors. (2, 22) While these platforms have had success in detecting specific compounds or chemical families, most are based on the binding of a single type of small molecule to a particular antibody or DNA aptamer, precluding broad detection of estrogenic activity (EA). Furthermore, antibodies can introduce cost and storage difficulties, and many platforms require analyte labeling with an electrochemical probe or fluorophore for detection.

Endocrine disrupting chemicals (EDCs) are increasingly identified as potent and pervasive risks to human health. They enter the environment through numerous human activities, including pesticide use, agriculture, and fracking, and they are found in consumer products such as plastic kitchen products and food can linings. (1-3) EDCs are especially dangerous because they are harmful at very low concentrations (picomolar to nanomolar), particularly to fetuses and newborns, (4-8) and they are implicated in increased occurrences of obesity, diabetes, infertility, and cancer. (9-11) The rapid and sensitive detection of these chemicals is therefore vital, ideally using equipment that is portable and inexpensive. Unfortunately, these compounds are particularly difficult to measure because they are not defined by a common chemical structure, but instead by their activity. (12, 13) To address this obstacle, we have developed a new detection paradigm for the sensitive, broad-spectrum detection of EDCs based on a native estrogen receptor alpha (ERα) construct expressed on the surface of. These engineered bacterial sensors enable the detection of many detrimental compounds as well as signal amplification from impedance measurements as they bind to modified electrodes. Rather than responding to individual components, this approach reports the total estrogenic activity of a sample using the biological receptor itself. Additional features of this sensing strategy include sample volumes of only 10 μL, rapid response rates, and the use of low-cost, disposable electrodes. As such, it is the first broad-spectrum EDC assay that is appropriate for field use.

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E. coli as a scaffold for the ERα protein resulted in significantly more sensitive measurements of E2 compared to the binding of ERα alone (E. coli cells to the gold surface, as compared to the significantly smaller free protein (E. coli or ERα added (E. coli were tested, and a dependence on the number of cells used for detection was observed (4/mL. The number of ERα proteins surface expressed on fresh E. coli was determined to be approximately 70,000 using a fluorescent coumarin–E2 conjugate,E. coli it was slightly lower (50,000/cell) (E. coli resulted in a small impedance response as compared to the lyophilized cells (E. coli killed with sodium azide to E. coli rendered nonviable, but alive and motile, by a low dose of chloramphenicol. The chloramphenicol-treated E. coli behaved as the untreated, live E. coli, and the sodium azide treated cells behaved similarly to the lyophilized cells (E. coli from a live sample were observed on electrodes by electron microscopy. The use of lyophilizedas a scaffold for the ERα protein resulted in significantly more sensitive measurements of E2 compared to the binding of ERα alone ( Figure 2 ). The enhanced sensitivity is due to a substantially increased impedance response from the recruitment of the largecells to the gold surface, as compared to the significantly smaller free protein ( Figure 2 b). Additionally, no signal change is observed in the presence of E2 but with noor ERα added ( Figure S3 ). Both fresh and lyophilizedwere tested, and a dependence on the number of cells used for detection was observed ( Figure S1 ). For lyophilized cells, the optimal number of cells was found to be 10/mL. The number of ERα proteins surface expressed on freshwas determined to be approximately 70,000 using a fluorescent coumarin–E2 conjugate, (26) while on the lyophilizedit was slightly lower (50,000/cell) ( Figure S2 ). This level of surface expression is expected, as the maximum number of ice nucleation proteins that were fused to ERα is on the order of 100,000. (27, 28) Freshresulted in a small impedance response as compared to the lyophilized cells ( Figure 2 b) likely due to their motility, which reduces their binding to the electrode surface. This hypothesis was supported by comparing detection withkilled with sodium azide torendered nonviable, but alive and motile, by a low dose of chloramphenicol. The chloramphenicol-treatedbehaved as the untreated, live, and the sodium azide treated cells behaved similarly to the lyophilized cells ( Figure S3 ). Consistent with this behavior, nofrom a live sample were observed on electrodes by electron microscopy.

Figure 2 Figure 2. Electrochemical sandwich assay for endocrine disrupting compounds (EDCs). (a) Nyquist plots of estradiol detection with the platform at concentrations ranging from 0 pM to 10 uM, along with the CPE fits used to determine the charge transfer radius (R CT ). (b) Estradiol concentration dependent R CT for ERα (blue), ERα on live E. coli (red), and ERα on lyophilized E. coli (black). Error bars represent the SD for n = 3 replicates.

R CT ) was derived from the CPE fits and was found to be proportional to the amount of ERα bound to the electrode and, therefore, the amount of substrate present. Using R CT as a proxy for the concentration of substrate, we were able to detect 500 pM E2 with a large linear range of detection up to 10 μM ( Detection of the binding event was accomplished with electrochemical impedance spectroscopy (EIS) in ferricyanide/ferrocyanide solution. This technique is rapid (providing readout in minutes), sensitive, and label-free. (29, 30) Nyquist plots were generated from each EIS scan performed, and the data were fit to a constant phase element (CPE) circuit model ( Figure 2 a). The charge transfer resistance () was derived from the CPE fits and was found to be proportional to the amount of ERα bound to the electrode and, therefore, the amount of substrate present. Usingas a proxy for the concentration of substrate, we were able to detect 500 pM E2 with a large linear range of detection up to 10 μM ( Figure 2 b). As the required sample volume is especially low (10 μL), we were able to detect femtomoles of estradiol at the detection limit.

50 values (shown as vertical lines). All agonists tested (4-NP, GEN, DES, and BPA) produced linear responses over an extended concentration range, with increasing R CT as EDC concentration increased. Some nonlinearity was observed at low concentrations of GEN, which could be due to complexities in the ternary complex formation. For DES, reduced linearity was observed as the detection limit was approached. Each of these compounds was detectable at exceptionally low concentrations, and most could be quantified below their IC 50 values. DES was detectable to concentrations ten times its IC 50 value. Unlike the EDCs that bind ERα, this platform shows no response to progesterone, indicating its specificity for estrogenic compounds. Similarly, this platform showed no response to the antagonist tamoxifen (TAM), indicating that the conformation of the ERα–antagonist complex does not bind the monobody on the electrode surface ( The system was found to be especially versatile, with detection of chemicals that have disparate chemical structures but similar bioactivity. The EDCs tested that bind ERα are 4-nonylphenol (4-NP), genistein (GEN), diethylstilbestrol (DES), and bisphenol A (BPA). Progesterone (P4) was used as a negative control, as P4 is not a substrate for ERα binding. In Figures 3 and 4 a (turquoise), each EDC was tested over a range of concentrations selected on the basis of their respective ICvalues (shown as vertical lines). All agonists tested (4-NP, GEN, DES, and BPA) produced linear responses over an extended concentration range, with increasingas EDC concentration increased. Some nonlinearity was observed at low concentrations of GEN, which could be due to complexities in the ternary complex formation. For DES, reduced linearity was observed as the detection limit was approached. Each of these compounds was detectable at exceptionally low concentrations, and most could be quantified below their ICvalues. DES was detectable to concentrations ten times its ICvalue. Unlike the EDCs that bind ERα, this platform shows no response to progesterone, indicating its specificity for estrogenic compounds. Similarly, this platform showed no response to the antagonist tamoxifen (TAM), indicating that the conformation of the ERα–antagonist complex does not bind the monobody on the electrode surface ( Figure S5 ).

Figure 3 Figure 3. Endocrine disrupting compound concentration dependent R CT for ERα on lyophilized E. coli with compounds that bind ERα: 4-nonylphenol (4-NP, top left), genistein (GEN, top right), and diethylstilbestrol (DES, bottom left). Progesterone, which does not bind ERα, shows no R CT response (bottom right). Error bars represent the SD for n = 3 replicates.

Figure 4 Figure 4. EDC detection from complex solutions. (a) Endocrine disrupting compound concentration dependent R CT for ERα on lyophilized E. coli with BPA in buffer (turquoise) and in infant formula (purple). (b) Combinations of BPA, 4-NP, DES, and GEN with comparable estradiol concentrations (black). Each solution contains 50% of one EDC, with 16.67% of each of the other three EDCs (where 100% would represent the known IC 50 concentration) . Samples with 50% BPA (dark blue), 50% 4-NP (turquoise), and 50% DES (green) are shown. (c) Estrogenicity of plastic (red) and glass (blue) baby bottles before and after microwaving. Error bars represent the SD for n = 3 replicates.

50 value), with 16.67% of each of the other three EDCs. The absolute concentrations of the components appear in R CT values for the combined EDCs were compared to the equivalent concentration of E2 as a measure of the EA of the solution. Independent of the ratio of EDCs in the solution, the R CT was found to be comparable to the equivalent concentration of E2 ( In contaminated systems, EDCs rarely occur as a single compound. Rather, they are often mixed, providing an aggregate effect. The combined interaction of all the EDCs present with the ERα protein yields a response that can be benchmarked as a concentration of the native substrate, E2, that would produce similar activity. This equivalent response is termed the “estrogenic activity” (EA) of the solution. The sensor was therefore evaluated for its ability to determine EA of complex mixtures. The EDCs previously measured (BPA, 4-NP, DES, and GEN) were combined and compared with comparable estradiol concentrations. Each solution contained 50% of one EDC (relative to its ICvalue), with 16.67% of each of the other three EDCs. The absolute concentrations of the components appear in Table S1 . Thevalues for the combined EDCs were compared to the equivalent concentration of E2 as a measure of the EA of the solution. Independent of the ratio of EDCs in the solution, thewas found to be comparable to the equivalent concentration of E2 ( Figure 4 b). This platform therefore shows the distinct advantage of providing a readout of the total EA from a complex mixture of components even when their specific identities are unknown.

50 value, despite the addition of protein, lipid, and small molecule components. Below this concentration, the signal was indistinguishable from background, likely due to surface passivation from proteins in the formula. Importantly, this approach enables detection of target compounds present in complex mixtures of proteins and small molecules. As EDCs are especially deleterious for proper development, their presence has been especially problematic in infant products. As one relevant example, the detection of BPA was evaluated in infant formula ( Figure 4 a). BPA was added to reconstituted formula from a commercial source in varying concentrations. The ability of the system to detect BPA was linear above the ICvalue, despite the addition of protein, lipid, and small molecule components. Below this concentration, the signal was indistinguishable from background, likely due to surface passivation from proteins in the formula.

As a final experiment, we evaluated the ability of the system to detect EA from an everyday source without prior knowledge of the contaminants. In the literature, E-SCREEN assays have shown that certain “BPA-free” plastic baby bottles release EDCs upon microwave heating. (31) We sought to replicate this experiment using the faster and lower volume electrochemical assay described herein. Prior to microwave heating the plastic bottle, the buffer had no observable EA. However, after microwaving for ten 2 min periods, the buffer in the plastic bottle had significant EA, comparable to 100 nM E2. In contrast, the buffer in a glass bottle contained no EA before or after microwaving ( Figure 4 c).

Through this work, we have developed a new approach for determining the estrogenic activity of endocrine disrupting compounds. By combining impedance spectroscopy based detection with the signal amplification provided by a lyophilized E. coli scaffold, large responses in the charge transfer resistance of the electrode are observed, even in the presence of sub-ppb estradiol. The system provides the first reported sensor that responds broadly to all EDCs, and since it is based on inexpensive disposable electrode technology, it can be used in the field. The 10 μL sample size is far smaller than that needed for cell-based growth assays, and the readout is available in minutes, not days. Furthermore, the application of lyophilized E. coli as a scaffold for our protein provides a new method of signal amplification, and is crucially important for reaching the low detection limits that these compounds require. The system also shows promising compatibility with complex sample matrices, such as infant formula. This new sensing approach should be applicable to other diverse families of compounds that bind to a single receptor, such as PPARγ, and current efforts in our laboratory are exploring these possibilities.