WO2018118724A1

WO2018118724A1 - Sensitive detection of chemical species using a bacterial display sandwich

Info

Publication number: WO2018118724A1
Application number: PCT/US2017/066901
Authority: WO
Inventors: Matthew B. Francis; Ariel L. Furst; Alexander C. HOEPKER
Original assignee: Regents Of The University Of California
Priority date: 2016-12-19
Filing date: 2017-12-18
Publication date: 2018-06-28

Abstract

Chemical analytes are detected by combining bacteria functionalized with a first receptor, an electrode functionalized with second receptor, and a medium comprising the analyte, under conditions wherein the bacteria specifically bind the electrode through a specific first receptor-analyte-second receptor complex, producing a detectable electrical signal at the electrode as in indication of the presence of the analyte.

Description

Sensitive Detection of Chemical Species Using A Bacterial Display Sandwich

Inventors: Matthew B. Francis, Ariel L. Furst, Alexander C. Hoepker, all of Berkeley, CA Applicant/ Assignee: The Regents of the University of California

Priority: Ser No. 62/436,351 ; Filed: Dec 19, 2016; Conf No. 1471

This invention was made with government support under Grant Numbers CHE-1413666 awarded by the National Science Foundation. The government has certain rights in the invention.

[001] Introduction

[002] 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 (ERa) construct expressed on the surface of E. coli. 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 μί, 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.

[003] The current standards for EDC detection are cell-based assays (originally the E- SCREEN assay,¹⁴ and, more recently, transactivation assays¹⁵'¹⁶ and yeast-based assays¹⁷'¹⁸) and radioactive¹⁹ 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 17 -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.

[004] Summary of the Invention

[005] The approach described herein is based on a novel electrochemical sandwich assay. One application involves the use of lyophilized bacteria, such as E. coli, to cause changes in the surface impedance upon binding. Several unique aspects of this strategy enable the detection of a range of analytes, such as estrogenic compounds, at exceptionally low concentrations. The bacterial surfaces are typically engineered to display a capture agent (e.g. ERa), which facilitates detection of any compounds that associate with its binding pocket.²³ The use of lyophilized bacterial advantageously limits their viability and increases storage life. Another component of the sandwich assay is an electrochemical working electrode modified with a receptor that binds

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to capture agent only when the analyte/ligand is present. ' In particular embodiments, a protein receptor, such as a monobody, may be attached through the interactions of a cysteine thiol with a disposable gold electrode surface. For example, the specificity of an electrode surface-bound monobody receptor observable by scanning electron microscopy of the working electrode surfaces. In the presence of estradiol (E2), E. coli were observed on the surface, while in the absence of E2, no E. coli bound the surface.

[006] Our invention provides a new platform for the sensitive detection of trace compounds using electrochemical methods. This technique relies on a pair of biomolecules that can form a ternary complex with analytes of interest. One of the biomolecules is attached to inexpensive commercial electrodes, and the other is displayed on the surface of bacterial cells. When the analyte of interest is present, the bacteria are recruited to the surface. This produces a large change in the electrochemical properties that can be measured. This system works with environmental samples, food, drink and water samples, product extracts, swipes, etc., physiological samples, including blood, urine, saliva, and other bodily samples, etc. and through selections of the binding groups detection platforms are readily applied to a broad range of analytes. We have demonstrated this system for the detection of endocrine-binding compounds that are commonly found in contaminated water supplies. In this embodiment, the ligand- binding domain of the human estrogen receptor was displayed on the bacterial surfaces. The bacteria were lyophilized and thus no longer viable. A monobody was displayed on the electrode surface. Low detection levels were obtained for estradiol, bisphenol A (BPA), diethylstilbestrol (DES), genistein (GEN), and 4-nonylphenol (4-NP).

[007] The invention provides methods and compositions for detecting chemical analytes in various media at risk of human contact. In an aspect the invention provides a method of detecting a chemical analyte, the method comprising: combining bacteria functionalized with a first receptor, an electrode functionalized with second receptor, and a medium comprising the analyte, under conditions wherein the bacteria specifically bind the electrode through a specific first receptor- analyte- second receptor complex, producing a detectable electrical signal at the electrode as in indication of the presence of the analyte.

[008] In embodiments:

[009] - the signal is quantitative with respect to the analyte;

[010] - the signal is a change in impedance;

[011] - the electrode produces a distinct signal when as few as 10 bacteria are bound;

[012] - the electrode produces a linear signal from between 10 to 1000 bound bacteria;

[013] - the analyte is at a concentration on the order of ppm, ppb or ppt;

[014] - the first and/or second receptor is a polynucleotide, polypeptide or polysaccharide;

[015] - the first receptor is recombinantly expressed by the bacteria;

[016] - the first receptor is expressed on the surface of the bacteria;

[017] - the bacteria are nonviable, dead, lyophilized, etc.;

[018] - the analyte is an endocrine disrupting chemical;

[019] - the first receptor is a promiscuous receptor, e.g. ERa, PPARy;

[020] - the second receptor is a monobody;

[021] - the medium comprises a mixture of different endocrine disrupting chemical, such as a mixture comprising 2, 3 or 4 of BPA, 4-NP, DES, and GEN, and the signal is indicative of the estrogenic activity (EA) of the medium; and/or

[022] - the medium is a human consumable or ingestible consumer produce or extract thereof.

[023] The invention provides reagents and kits for practicing the disclosed methods.

[024] The invention encompasses all combination of the particular embodiments recited herein, as if each combination had been laboriously recited, such as wherein the analyte is an endocrine disrupting chemical, the first receptor is ERa, and the second receptor is an ERa- estradiol selective monobody protein. [025] Description of Particular Embodiments of the Invention

[026] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms "a" and "an" mean one or more, the term "or" means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones described herein.

[027] The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[028] Example: Quantifying hormone disrupters with an engineered bacterial biosensor

[029] Abstract: Endocrine disrupting compounds are found in increasing amounts in our environment, originating from pesticides, plasticizers, and pharmaceuticals, among other sources. Although the full impact of these compounds is still under study, they have already been implicated in diseases such as obesity, diabetes, and cancer. The list of chemicals that disrupt normal hormone function is growing at an alarming rate, making it crucially important to find sources of contamination and identify new compounds that display this ability. However, there is currently no broad-spectrum, rapid test for these compounds, as they are difficult to monitor because of their high potency and chemical dissimilarity. To address this, we have developed new compound detection strategy that is both fast and portable, and it requires no specialized skills to perform. We exemplify the system with a native estrogen receptor construct expressed on the surface of E. coli, which enables both the detection of many detrimental compounds and signal amplification from impedance measurements due to the binding of bacteria to a modified electrode. With this approach, sub-ppb levels of estradiol and ppm levels of bisphenol A are detected in complex solutions. Rather than responding to individual components, this system reports the total estrogenic activity of a sample using the most relevant biological receptor. As an applied example, estrogenic chemicals released from a plastic baby bottle following microwave heating were detectable with this technique. This system is broadly applicable to the detection of chemically diverse classes of compounds that bind to a single receptor.

[030] Results and Discussion

[031] The use of lyophilized E. coli as a scaffold for the ERa protein resulted in significantly more sensitive measurements of E2 compared to the binding of ERa alone. The enhanced sensitivity is due to a substantially increased impedance response from the recruitment of the large E. coli cells to the gold surface, as compared to the significantly smaller free protein. Additionally, no signal change is observed in the presence of E2 but with no E. coli or ERa added. Both fresh and lyophilized E. coli were tested, and a dependence on the number of cells used for detection was observed. For lyophilized cells, the optimal number of cells was found to be 10⁴/mL. The number of ERa proteins surface expressed on fresh E. coli was determined to be approximately 70,000 using a fluorescent coumarin-E2 conjugate,²⁶ while on the lyophilized E. coli it was slightly lower (50,000/cell). This level of surface expression is expected, as the maximum number of ice nucleation proteins that were fused to ERa is on the order of

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100,000. ' Fresh E. coli resulted in a small impedance response as compared to the lyophilized cells likely due to their motility, which reduces their binding to the electrode surface. This hypothesis was supported by comparing detection with 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. Consistent with this behavior, no E. coli from a live sample were observed on electrodes by electron microscopy.

[032] 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. The charge transfer resistance (Rcr) was derived from the CPE fits and was found to be proportional to the amount of ERa bound to the electrode and, therefore, the amount of substrate present. Using RCT 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 μΜ. As the required sample volume is especially low (10 μ > we were able to detect femtomoles of estradiol at the detection limit.

[033] 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 ERa 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 ERa binding. Each EDC was tested over a range of concentrations selected based on their respective IC₅₀ values. 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. Each of these compounds was detectable at exceptionally low concentrations, and generally could be quantified below their IC₅₀ values. DES was detectable to concentrations ten times its IC₅₀ value. Unlike the EDCs that bind ERa, 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 ERa-antagonist complex does not bind the monobody on the electrode surface.

[034] 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 ERa 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 IC₅₀ value), with 16.67% of each of the other three EDCs. We determined the absolute concentrations of the components. The RCT 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 RCT was found to be comparable to the equivalent concentration of E2. 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.

[035] 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. 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 IC₅₀ value, despite the addition of protein, lipid and small molecule components.

[036] We also 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 two-minute 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.

[037] 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 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 is applicable to other diverse families of compounds that bind to a single receptor, such as PPARy.

[038] References:

[039] 1. Melnick, R.; Lucier, G.; Wolfe, M.; Hall, R.; Stancel, G.; Prins, G.; Gallo, M.; Reuhl, K.; Ho, S.-M.; Brown, T.; Moore, J.; Leakey, J.; Haseman, J.; Kohn, M. Summary of the National Toxicology Program' s report of the endocrine disrupters lowdose peer review.

Environ. Health Perspect. 2002, 110, All- 431.

[040] 2. Petrovic, M.; Eljarrat, E.; Lopez de Alda, M. J.; Barcelo, D. Endocrine disrupting compounds and other emerging contaminants in the environment: A survey on new monitoring strategies and occurrence data. Anal. Bioanal. Chem. 2004, 378, 549-562.

[041] 3. Welshons, W. V.; Nagel, S. C; vom Saal, F. S. Large effects from small exposures. III. Endocrine mechanisms mediating effects of bisphenol A at levels of human exposure.

Endocrinology 2006, 147, S56 -S69.

[042] 4. Vandenberg, L. N.; Colborn, T.; Hayes, T. B.; Heindel, J. J.; Jacobs Jr., D. R.; Lee, D. H.,; Shoida, T.; Soto, A. M.; vom Saal, F. S.; Welshons, W. V.; Zoeller, R. T.; Myers, J. P. Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses. Endocrine Rev. 2012, 33, 378 - 455.

[043] 5. Balabanic, D.; Rupnik, M.; Klemencic, A. K. Negative impact of endocrine- disrupting compounds on human reproductive health. Reproduction, Fertility and

Development 2011, 23, 403-416.

[044] 6. Colborn, T.; vom Saal, F. S.; Soto, A. M. Developmental effects of endocrine- disrupting chemicals in wildlife and humans. Environ. Health Perspect. 1993, 101, 378-384.

[045] 7. McLachlan, J. A. Environmental signaling: What embryos and evolution teach us about endocrine disrupting chemicals. Endocrine Rev. 2001, 22, 319-341.

[046] 8. Schug, T. T.; Johnson, A. F.; Birnbaum, L. S.; Colborn, T.; Guilette Jr., L. J.; Crews, D. P.; Collins, T.; Soto, A. M.; vom Saal, F. S.; McLachlan, J. A.; Sonnenschein, C; Heindel, J. J. Minireview: endocrine disruptors: past lessons and future directions. Mol. Endocrinol. 2016, 30, 833- 847. [047] 9. Newbold, R.; Padilla-Banks, E.; Jefferson, W.; Heindel, J. Effects of endocrine disrupters on obesity. Int. J. Androl. 2008, 31, 201-208.

[048] 10. Roy, J. R.; Chakraborty, S.; Chakraborty, T. R. Estrogen-like endocrine disrupting chemicals affecting puberty in humans-a review. Med. Sci. Monit. 2009, 15, RA137-145.

[049] 11. Birnbaum, L. S.; Fenton, S. E. Cancer and developmental exposure to endocrine disrupters. Environ. Health Perspect. 2003, 111, 389-394.

[050] 12. Blair, R. M.; Fang, H.; Branham, W. S.; Hass, B. S.; Dial, S. L.; Moland, C. L.; Tong, W.; Shi, L.; Perkins, R.; Sheehan, D. M. The Estrogen Receptor Relative Binding Affinities of 188 Natural and Xenochemicals: Structural Diversity of Ligands. Toxicol. Sci. 2000, 54, 138-153.

[051] 13. Matthews, J.; Celius, T.; Halgren, R.; Zacharewski, T. Differential estrogen receptor binding of estrogenic substances: a species comparison. /. of Steroid Biochem. & Molec. Biol. 2000, 74, 223-234.

[052] 14. Soto, A. M.; Sonnenschein, C; Chung, K. L.; Fernandez, M. F.; Olea, N.; Serrano, F. O. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic

environmental pollutants. Environ. Health Perspect. 1995, 103, 113-122.

[053] 15. Paech, K.; Webb, P.; Kuiper, G. G. J. M.; Nilsson, S.; Gustafsson, J.-A.; Kushner, P. J.; Scanlan, T. S. Differential Ligand Activation of Estrogen Receptors ERa and ER at API Sites. Science 1997, 277, 1508-1510.

[054] 16. Andersen, H. R.; Vinggaard, A. M.; Rasmussen, T. H.; Gjermandsen, I. M.;

Bonefeld- J0rgensen, E. C. Effects of Currently Used Pesticides in Assays for Estrogenicity, Androgenicity, and Aromatase Activity in Vitro. Toxicol. Appl. Pharmacol. 2002, 179, 1-12.

[055] 17. Kolle, S. N.; Kamp, H. G.; Huener, H. A.; Knickel, J.; Verlohner, A.; Woitkowiak, C; Landiedel, R.; van Ravenzwaay, B. In house validation of recombinant yeast estrogen and androgen receptor agonist and antagonist screening assays. Toxicol. In Vitro 2010, 24, 2030- 2040.

[056] 18. Beresford, N.; Routledge, E. J.; Harris, C. A.; Sumpter, J. P. Issues arising when interpreting results from an in vitro assay for estrogenic activity. Toxicol. Appl. Pharmacol. 2000, 162, 22 - 33.

[057] 19. Kinnberg, K. Evaluation of in vitro assays for determination of estrogenic activity in the environment. Working Report No. 43, 2003. http:/www2.mst.dk/udgiv/publications/2003/87- 7972-922-3/pdf/87-7972-923-l.pdf. Accessed 10/18/16.

[058] 20. Bolger, R.; Wiese, T. E.; Ervin, K.; Nestich, S.; Checovich, W. Rapid screening of environmental chemicals for estrogen receptor binding capacity. Envorin. Health Perspect. 1998, 106, 551-557. [059] 21. Varriale, A.; Pennacchio, A.; Pinto, G.; Oliviera, G.; D'Errico, S.; Majoli, A.; Scala, A.; Capo, A.; Pennacchio, A.; Di Giovanni, S.; Staiano, M.; D'Auria, S. A Fluorescence Polarization Assay To Detect Steroid Hormone Traces in Milk. /. Agric. Food Chem. 2015, 63, 9159-9164.

[060] 22. Scognamiglio, V.; Antonacci, A.; Patrolecco, L.; Lambreva, M. D.; Litescu, S. C; Ghuge, S. A.; Rea, G. Analytical tools monitoring endocrine disrupting chemicals. TrAC. 2016, 80, 555-567.

[061] 23. Pillon, A.; Boussioux, A.-M.; Escande, A.; Ai^'t-Ai^'ssa, S.; Gomez, E.; Fenet, H.; Ruff, M.; Moras, D.; Vignon, F.; Duchesne, M.-J.; Casellas, C; Nicolas, J. C; Balaguer, P. Binding of estrogenic compounds to recombinant Estrogen Receptor-a: Application to environmental analysis. Environ. Health Perspect. 2005, 113, 278-284.

[062] 24. Koide, A.; Abbatiello, S.; Rothgery, L.; Koide, S. Probing protein conformational changes in living cells by using designer binding proteins: Application to the estrogen receptor. Proc. Natl. Acad. Sci. 2002, 99, 1253-1258.

[063] 25. Huang, J.; Koide, A.; Nettle, K. W.; Greene, G. L.; Koide, S. Conformation-specific affinity purification of proteins using engineered binding proteins: application to the estrogen receptor. Protein Expr. Purif. 2006, 47, 348-354.

[064] 26. MacDonald, J. I.; Munch, H. K.; Moore, T.; Francis, M. B. One-step site-specific modification of native proteins with 2-pyridinecarboxaldehydes. Nat. Chem. Biol. 2015, 11, 326- 331.

[065] 27. van Bloois, E.; Winter, R. T.; Kolmar, H.; Fraaije, M. W. Decorating microbes: surface display of proteins on Escherichia coli. Trends in Biotechnol. 2009, 29, 79-86.

[066] 28. Li, L.; Kang, D. G.; Cha, H. J. Functional display of foreign protein on surface of Escherichia coli using N-terminal domain of Ice Nucleation Protein. Biotechnol. and

Bioengineer. 2004, 85, 214-221.

[067] 29. Lisdat, F.; Schaefer, D. The use of electrochemical impedance spectroscopy for biosensing. Anal. Bioanal. Chem. 2008, 391, 1555-1567.

[068] 30. Guo, X.; Kulkarni, A.; Doepke, A.; Halsall, H. B.; Iyer, S.; Heineman, W. R.

Carbohydrate-based label-free detection of Escherichia coli ORN 178 using Electrochemical Impedance Spectroscopy. Anal. Chem. 2012, 84, 241-246.

[069] 31. Yang, C. Z.; Yaniger, S. I.; Jordan, V. C; Klein, D. J.; Bittner, G. D. Most plastic products release estrogenic chemicals: a potential health problem that can be solved. Environ. Health Perspect. 2011, 119, 989-996.

Claims

CLAIMS:

1. A method of detecting a chemical analyte, the method comprising:

combining bacteria functionalized with a first receptor, an electrode functionalized with second receptor, and a medium comprising the analyte, under conditions wherein the bacteria specifically bind the electrode through a specific first receptor-analyte-second receptor complex, producing a detectable electrical signal at the electrode as in indication of the presence of the analyte, wherein the signal is quantitative with respect to the analyte.

2. The method of claim 1 wherein the signal is a change in impedance.

3. The method of any of claims 1-2 wherein the electrode produces a distinct signal when 10 bacteria are bound.

4. The method of any of claims 1-3 wherein the electrode produces a linear signal from between 10 to 1000 bound bacteria.

5. The method of any of claims 1-4 wherein the analyte is at a concentration on the order of ppm, ppb or ppt.

6. The method of any of claims 1-5 wherein the first and/or second receptor is a polynucleotide, polypeptide or polysaccharide.

7. The method of any of claims 1-6 wherein the first receptor is recombinantly expressed by the bacteria.

8. The method of any of claims 1-7 wherein the first receptor is expressed on the surface of the bacteria.

9. The method of any of claims 1-8 wherein the bacteria are nonviable, dead, lyophilized, etc.

10. The method of any of claims 1-9 wherein the analyte is an endocrine disrupting chemical.

11. The method of any of claims 1-10 wherein the first receptor is a promiscuous receptor, e.g. ERa, PPARy.

12. The method of any of claims 1-11 wherein the second receptor is a monobody.

13. The method of any of claims 1-12 wherein the medium is a human consumable or ingestible consumer product or extract thereof.

14. The method of any of claims 1-13 wherein the analyte is an endocrine disrupting chemical, the first receptor is ERa, and the second receptor is an ERa-estradiol selective monobody protein.

15. The method of any of claims 1-14 wherein the medium comprises a mixture of different endocrine disrupting chemical, and the signal is indicative of the estrogenic activity (EA) of the medium.