Biosensor for rapid detection of per- and polyfluoroalkyl substances (PFAS)

Abstract

Disclosed herein is a biosensor fusion protein comprising an N-terminal half of a human liver fatty acid binding protein (hLFABP) and a C-terminal half of the hLFABP separated by a circularly permuted fluorescent protein (cpFP), wherein the binding of the N-terminal half of the hLFABP and the C-terminal half of the of the hLFABP to a per- or polyfluoroalkyl substance (PFAS) elicits a change in fluorescence of the cpFP. Also disclosed method for detecting per- and polyfluoroalkyl substances (PFAS) in a sample, comprising contacting the sample with the biosensor disclosed herein, assaying the sample for fluorescence, wherein an increase in fluorescence is an indication of the presence of PFAS. In some embodiments, the PFAS is perfluorooctanoic acid (PFOA). In some embodiments, the sample is a water sample.

Description

BIOSENSOR FOR RAPID DETECTION OF PER- AND POLYFLUOROALKYL SUBSTANCES (PFAS)CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 582,124, filed September 12, 2023, which is hereby incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] This application contains a sequence listing filed in ST.26 format entitled “222117-2380 Sequence Listing” created on August 21 , 2024, and having 14,126 bytes. The content of the sequence listing is incorporated herein in its entirety.BACKGROUND OF THE INVENTION

[0003] Per and poly-fluoroalkyl substances (PFAS) are a group of man-made chemicals that have become an urgent public health concern. Referred to as “forever chemicals” by news and media outlets, these fluorochemicals are pervasive environmental contaminants without a robust natural degradation pathway (Buck, R. C., et al. Integr Environ Assess Manag. 2011 7:513-541 ; Wang, Z., et al. Environ. Sci. Technol. 2021 55:15575-15578). Due to their unique chemical composition, PFAS are water and / or oil resistant which makes them advantageous to use in a wide number of commercial applications. First manufactured in the 1940s, PFAS have been used in a variety of industrial and commercial products including fire-fighting foams, synthetic fabrics, medical devices, food packaging, and cosmetic goods (D’Agostino, L. A., et al. Environ Toxicol Chem. 2017 36:2012-2021; Dauchy, X., et al. Chemosphere. 2017 183:53-61 ; Fenton, S. E., et al. Environmental Toxicology and Chemistry. 2021 40:606-630; Wang, Z., et al. Environ. Sci. Technol. 2017 51 :2508-2518). PFAS consist of chains of highly fluorinated carbon atoms bound to polar head groups which are typically carboxylic acids, tertiary amines, or sulfide moieties (Buck, R. C., et al. Integr Environ Assess Manag. 2011 7:513-541; Harding- Marjanovic, K. C., et al. Environ. Sci. Technol. 2015 49:7666-7674). Over 3000 different types of PFAS have been industrially manufactured with two major categories consisting of perfluorocarboxylic acids (PFCAs) and perfluorosulfonic acids (PFSAs) (Buck, R. C., et al. Integr Environ Assess Manag. 2011 7:513-541). Unfortunately, their amphiphilic properties confer high solubility under aqueous conditions, causing these chemicals to be a prevalent, mobile and persistent set of environmental contaminants (Ahrens, L., et al. Environ Toxicol Chem. 2014 33:1921-1929; Arvaniti, O. S., et al. Science of The Total Environment. 2015 524- 525:81-92; Brunn, H., et al. Environ Sci Eur. 2023 35:20).

[0004] Despite only having been manufactured for less than a century, most residents of industrialized countries have been exposed to PFAS (Kato, K., et al. Chemosphere. 2018 209:338-345; Kato, K., et al. Environ. Sci. Technol. 2011 45:8037-8045; Olsen, G. W., et al. Environmental Research. 2017 157:87-95). Among a representative sample of the U.S. population, 95% of human serum analyses yielded a positive result for PFAS (Calafat, A. M., et al. Environ. Sci. Technol. 200640:2128-2134; Calafat, A. M., et al. Environ. Sci. Technol. 2007 41 :2237-2242). With a half-life of several years, and the inability to naturally degrade, PFAS can accumulate in human tissues through long-term exposure, even with a source containing relatively low concentrations (Fenton, S. E., et al. Environmental Toxicology and Chemistry. 2021 40:606-630; Post, G. B., et al. Environmental Research. 2012 116:93-117). While new toxicological effects are continuing to be discovered, long-term exposure to PFAS, specifically medium chain perfluoroalkyl acids like PFOA, have been linked to numerous health problems including increased cholesterol levels (Lin, P.-l. D., et al. Environment International. 2019 129:343-353; Steenland, K., et al. Environ Health Perspect. 2010 118:1100-110; Steenland, K., et al. American Journal of Epidemiology. 2009 170:1268-1278), various cancers (Imir, O. B., et al. Nutrients. 2021 13:3902; Law, H., et al. Cancer Epidemiology. 2023 82:102296; Messmer, M. F., et al. Environ Health Insights. 2022 16:117863022210767), and reproductive issues (Chambers, W. S., et al. Front. Toxicol. 2021 3:732436). Many reviews summarizing toxicity and health information have been published (Fenton, S. E., et al. Environmental Toxicology and Chemistry. 2021 40:606-630; Chambers, W. S., et al. Front. Toxicol. 2021 3:732436; Boyd, R. I., et al. Cancers. 2022 14:2919; Stanifer, J. W., et al. CJASN. 2018 13:1479-1492; Steenland, K., et al. Environmental Research. 2021 194:110690). Furthermore, biomonitoring studies in a variety of species have shown that upon accumulation, the highest concentrations of PFOA to be found in the blood plasma and liver (Houde, M., et al. Environ. Sci. Technol. 2011 45:7962- 7973; Ng, C. A., et al. Environ. Sci. Technol. 2015 49:12306-12314). This has since been further elucidated as PFOA is shown to bind to relevant proteins including liver fatty acid binding protein (LFABP) and serum albumin (Bischel, H. N., et al. Environ. Sci. Technol. 2010 47:11293-11301).

[0005] With a rise in evidence of PFAS accumulation and toxicity comes a wave of regulatory changes and calls for action that highlight the necessity of quick, relatively easy ways to detect chemicals like PFOA. However, this challenge has proven non-trivial given the diversity of the chemicals as well as their limited reactivity and vast concentrations ranges. Currently, standard PFAS detection relies on chromatography techniques coupled with tandem mass spectroscopy. These methods are highly precise with detection limits in the range of 1ng / L (1 ppt) for aqueous samples (EPA Methods 533, 537, and 537.1). Increasing health concerns and new regulations in response to these concerns have led to development of new PFAS sensors capable of detecting compounds in drinking water. While still constrained to bench level research, the most successful technologies revolve around the use of molecularly imprinted polymers (MIPS) to capture and detect PFAS down to the ppt levels determined in health advisories set by the EPA (Feng, H., et al. Sensors and Actuators B: Chemical. 2014 195:266-273; Jiao, Z., et al. Microchim Acta. 2018 185:473; Pitruzzella, R., et al. Chemosensors. 2023 11 :211). Nonetheless, these methods often require extensive sample preparation, and / or are limited to drinking water samples making PFAS detection impractical for other applications and inaccessible for most communities.

[0006] Human exposure to PFAS has been contributed to multiple pathways including ingestion of contaminated water and food including crops grown in contaminated soils and biosolids as well as general dermal adsorption (De Silva, A. O., et al. Environ Toxicol Chem. 2021 40:631-657). Therefore, as PFAS contamination is found to be more and more ubiquitous in the environment, the necessity of detection at these higher concentrations and in a vast variety of matrices becomes clearer. In fact, several states in the U.S. report PFOA and PFOS concentrations in the part per million (ppm) in non-drinking water and various soil sources (Andrews, D. Q., et al. AWWA Water Science. 2021 10.1002 / aws2.1252; Carlson, G. L, et al. Chemosphere. 2020 261 :128078). High levels of PFAS pollution are common near manufacturing facilities, sites storing PFAS related waste, and areas utilizing fluorinated AFFFs like airports (Salvatore, D., et al. Environ. Sci. Technol. Lett. 2022 9:983-990). In fact, limited analyses of ground water and sediment samples in locations where PFAS containing AFFs were used, have demonstrated that a wide variety of PFAS chemicals can persist in the environment at high concentrations, several decades after release (Dauchy, X., et al. Chemosphere. 2017 183:53-61 ; Dauchy, X., et al. Chemosphere. 2019 214:729-737; Guelfo, J. L., et al. Environ. Sci. Technol. 201347:4164-4171 ; Houtz, E. F., et al. Environ. Sci. Technol. 2013 47:8187-8195; Reinikainen, J., et al. Science of The Total Environment. 2022 829:154237).

[0007] Agricultural lands have become reservoirs for PFAS as these chemicals are emitted directly into the environment or brought in through irrigation waters and soil amendments like treated sewage sludge and soil conditioners (Brunn, H., et al. Environ Sci Eur. 2023 35:20; Ghisi, R., et al. Environmental Research. 169:326-341 ; Stahl, T., et al. J. Agric. Food Chem. 201866:10147-10153). Therefore, this contamination is reflected in food crops. Studies have shown plants are capable of taking up and accumulating PFAS with preference tomedium and short chain chemicals like PFOA and PFOS (Perez, F., et al. Environmental Research. 2014 135:181-189; Wang, W., et al. Chemosphere. 2020261:127584). Specifically, for highly contaminated areas, this has been shown to range from .g / kg to mg / kg dry weight levels in a variety of crops (Blaine, A. C., et al. Environ. Sci. Technol. 2014 48:14361-14368; Liu, Z., et al. Environment International. 2019 127:671-684; Zhu, H., et al. Science of The Total Environment. 2019 647:954-961), which is orders of magnitude above the limits set for drinking water. With this in mind, there is a clear need for easy and rapid PFAS detection in a multitude of matrices and in a wide range of concentrations with minimal pre-processing. In order to grow necessary food crops and livestock, and allow safe use of outdoor recreation areas, people must be able to easily determine contamination.

[0008] Biosensors have often been used for detection of pathogens and contaminants in agricultural products and environmental samples as they offer the advantage of tunability in terms of sensitivity and selectivity as well as the possibility for minimal sample pre-treatment (Karadurmus, L., et al. Food Measure. 2021 15:4582-4595; Mehrotra, P. J Oral Biol Craniofac Res. 2016 6:153-159; Nigam, V. K., et al. Journal of Microbiology and Biotechnology. 2015 25:1773-1781 ; Sahu, S., et al. ACS Sens. 2022 7:704-715). Despite not having been assessed for real world feasibility, several biosensor platforms have been developed for PFAS detection. Some of these technologies are considered “whole cell biosensors”, created around bacteria with engineered biological promoters that induce fluorescent protein expression upon PFAS interaction (Sunantha, G., et al. Science of The Total Environment. 2021 759:143544; Young, N. A., et al. Mil Med. 2021 186:801-807). However, these have yet to be optimized for quick read-outs, often taking 24-48 hours. Biosensors utilizing individual binding proteins and antibodies as PFAS receptors rather than whole cell systems, have also been shown to detect PFOA and / or PFOS with various transducers including fiber optics (Cennamo, N., et al. Taianta. 2018 178:955-961 ; Moro, G., et al. Results in Optics. 2021 5:100123). An acrylodan based fluorescent sensor was previously designed for detection of several PFAS in water based on human liver fatty acid binding protein (hLFABP) (Mann, M. M., et al. Biotech & Bioengineering. 2022 119:513-522). While promising, to utilize the robustness and ease of whole-cell biosensors for direct detection in a variety of solid sample formats, it is advantageous to develop a genetically encoded system or protein capable of intrinsic detection of PFAS.SUMMARY OF THE INVENTION

[0009] A fluorescent biosensor was engineered for the rapid detection of PFOA based on human liver fatty acid binding protein (hLFABP). By conjugating circularly permuted green fluorescent protein (cp.GFP) to a split hLFABP construct, the biosensor was able to detectperfluorooctanoic acid PFOA in PBS as well as environmental water samples with LODs of 235 and 335 ppb respectively. Furthermore, E. coli cells cytosolically expressing the protein-based sensor were demonstrated to quickly detect PFOA, demonstrating feasibility of whole-cell sensing in solid samples.

[0010] Disclosed herein is a biosensor fusion protein comprising an N-terminal half of a human liver fatty acid binding protein (hLFABP) and a C-terminal half of the hLFABP separated by a circularly permuted fluorescent protein (cpFP), wherein the binding of the N-terminal half of the hLFABP and the C-terminal half of the of the hLFABP to a per- or polyfluoroalkyl substance (PFAS) elicits a change in fluorescence of the cpFP.

[0011] In some embodiments, the N-terminal half of hLFABP comprises amino acids 1- 56 of the hLFABP, and the C-terminal half of hLFABP comprises amino acids 57-128 of the hLFABP.

[0012] In some embodiments, the fusion protein has the formula:N-hLFABP -1- FP-C -2- N-FP -3- hLFABP-C wherein “N-hLFABP” represents the N-terminal half of the hLFABP; wherein “hLFABP-C” represents the C-terminal half of the hLFABP; wherein “FP-C” represents an N-terminal half of the cpFP; wherein “N-FP” represents a C-terminal half of the cpFP; and wherein “-1-”, “-2-”, and “-3-” represents a first, second, and third flexible linker, respectively.

[0013] In some embodiments, the cpFP is a circularly permuted green fluorescent protein (cpGFP). For example, in some embodiments, the cpGFP comprises amino acids 147- 238 of GFP linked to amino acids 1-146 of GFP by the second flexible linker.ln some embodiments, the second flexible linker has the amino acid sequence GGTGGS (SEQ ID NO:7), GGSGGS (SEQ ID NO:8), or GGTGGSGGTGGS (SEQ ID NO:9). Therefore, in some embodiments, the cpGFP has the amino acid sequence SEQ ID NO:1.

[0014] In some embodiments, the N-terminal half of hLFABP has the amino acid sequence SEQ ID NO:2, and the C-terminal half of hLFABP has the amino acid sequence SEQ ID NO:3. In some embodiments, the first and / or third flexible linker has the amino acid sequence GG, GSG, or GGSGGS (SEQ ID NO:8). Therefore, in some embodiments, the biosensor has the amino acid sequence SEQ ID NO:4.

[0015] Also disclosed method for detecting per- and polyfluoroalkyl substances (PFAS) in a sample, comprising contacting the sample with the biosensor disclosed herein, assaying the sample for fluorescence, wherein an increase in fluorescence is an indication of the presence ofPFAS. In some embodiments, the PFAS is perfluorooctanoic acid (PFOA). In some embodiments, the sample is a water sample.

[0016]

[0017] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF FIGURES

[0018] Figures 1A to 1C show cpGFP.hLFABP construct design overview. Figure 1A shows linear representation of construct with linkers L1 (GSG) L2 (GGTGGS, SEQ ID NO:7) and L3 (GG). Figure 1B is a schematic of circularly permuted GFP.hLFABP at the protein level depicting binding of PFAS. Figure 1C is an overlay of apo form and holo form of wild type hLFABP complexed with palmitic acid (PBD IDs 3STN and 3STK respectively). Location of receptor splitting was partly based on perturbations at residues 56 and 57.

[0019] Figure 2 shows SDS-PAGE analysis of cpGFP.hLFABP purification via Ni-NTA resin. Lane M: protein marker, Lanes 1-6: purification fractions containing increasing concentrations of imidazole (50, 75, 100, 125, 200, and 500mM respectively). cpGFP.hLFABP is shown as a band of approximately 46.6 kDa.

[0020] Figure 3A contains representative absorption spectra for cpGFP.hLFABP (2pM). Spectral changes upon increased PFOA addition show a decreased and increased trend in intensity at 395nm and 485nm respectively. Figures 3B-3C contain representative fluorescence emission spectra for cpGFP.hLFABP (1uM) after excitation at 395nm (FIG. 3B) and 485nm (FIG. 3C). Spectral changes upon PFOA titration shows a decreased and increased overall intensity for excitation at 395nm and 485nm respectively. All curves are smoothed for qualitative analysis.

[0021] Figure 4 shows binding of PFOA to cpGFP.hLFABP (1 pM) after titration in PBS (black circles), creek water (squares), and PBS containing 1 pM SDS (triangles). Data are fitted to a one site binding model with the fractional occupancy represented as the fractional change in fluorescence intensity at 510nm after excitation at 485nm. The represented points are mean values ±SEM with n=3. Bottom table displays calculated model constants and limits of detection in all systems.

[0022] Figure 5: Fluorescence intensity change at 510nm after excitation at 485nm of E. coli expressing cpGFP.hLFABP upon PFOA titration. Data are normalized by cell concentration (OD600) and fitted to a log-dose response model. The represented points are mean values ±SEM with n=3.DETAILED DESCRIPTION

[0023] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0024] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0026] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0027] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0028] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0029] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0030] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.Definitions

[0031] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0032] “Fluorescent protein” refers to any protein capable of emitting light when excited with appropriate electromagnetic radiation. Fluorescent proteins include proteins having amino acid sequences that are either natural or engineered (e.g., green fluorescent protein (GFP), circularly permuted green fluorescent protein (cpGFP), and variants and derivatives thereof).

[0033] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. The term “polypeptide” encompasses naturally occurring or synthetic molecules. In addition, as used herein, the term “polypeptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type ofmodification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent crosslinking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins - Structure and Molecular Properties 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).

[0034] As used herein, “peptidomimetic” means a mimetic of a peptide which includes some alteration of the normal peptide chemistry. Peptidomimetics typically enhance some property of the original peptide, such as increase stability, increased efficacy, enhanced delivery, increased half life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Patent Nos. 5,631 ,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. One embodiment of the present invention is a peptidomimetic wherein the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic. Some non-limiting examples of unnatural amino acids which may be suitable amino acid mimics include p-alanine, L-a-amino butyric acid, L-y- amino butyric acid, L-a-amino isobutyric acid, L-e-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, N-s-Boc-N-a-CBZ-L-lysine, N-e-Boc-N-a-Fmoc-L-lysine, L- methionine sulfone, L-norleucine, L-norvaline, N-a-Boc-N-bCBZ-L-ornithine, N-5-Boc-N-a-CBZ- L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

[0035] The term “percent (%) sequence identity” or “homology” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriateparameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0036] A “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.Split hLFABP

[0037] Disclosed herein is a biosensor involving a split human liver fatty acid binding protein (hLFABP) that activates a circularly permuted fluorescent protein (cpFP) when it binds per- and polyfluoroalkyl substances (PFAS). In some embodiments, the biosensor involves an N-terminal half of a hLFABP and a C-terminal half of the hLFABP separated by a cpFP, wherein the binding of the N-terminal half of the hLFABP and the C-terminal half of the of the hLFABP to a per- or polyfluoroalkyl substance (PFAS) elicits a change in fluorescence of the cpFP.

[0038] In certain embodiments, the hLFABP comprises a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence MSFSGKYQLQSQENFEAFMKAIGLPEELIQKGKDIKGVSEIVQNGKHFKFTITAGSKVIQNEFTV GEECELETMTGEKVKTVVQLEGDNKLVTTFKNIKSVTELNGDIITNTMTLGDIVFKRISKRIL (SEQ ID NO: 10), or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. In some embodiments, the hLFABP is split at amino acids 56 and 57. Therefore, in some embodiments, the N-terminal half of the hLFABP has the amino acid sequence SEQ ID NO:2, or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:2, and the C- terminal half of the hLFABP has the amino acid sequence SEQ ID NO:3, or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including anypercent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:3.Circularly Permuted Fluorescent Protein

[0039] The disclosed biosensor can be constructed with any circularly permuted fluorescent protein. The choice of a particular circularly permuted fluorescent protein for use in a disclosed biosensor may depend on the desired emission spectrum for detection, and includes, but is not limited to, circularly permuted fluorescent proteins with green, blue, cyan, yellow, orange, red, or far-red emissions. Exemplary circularly permuted fluorescent proteins that can be used in the disclosed biosensor include circularly permuted green fluorescent protein (cpGFP), circularly permuted superfolder GFP (cpsfGFP), circularly permuted mApple (cpmApple), circularly permuted mCherry (cpmCherry), circularly permuted mKate (cpmKate), circularly permuted enhanced green fluorescent protein (cpEGFP), circularly permuted Venus (cpVenus), circularly permuted Citrine (cpCitrine), and circularly permuted enhanced yellow fluorescent protein (cpEYFP). The process of circular permutation involves fusion of natural termini through the addition of a flexible peptide linker to create new termini near the chromophore(Topell, Hennecke, and Glockshuber 1999). This structure imparts greater mobility and often greater versatility of spectral properties. (Topell, Hennecke, and Glockshuber 1999) Many fluorescent proteins share rigid beta barrel structures in which circular permutation is achievable by utilizing break points found in flexible loop regions connecting p sheets. In fact, circular permutation has been utilized in a multitude of fluorescent proteins including: GFP (Topell, Hennecke, and Glockshuber 1999), sfGFP (Campbell et al. 2022), mApple (Zhao et al. 2011), mCherry (Shui et al. 2011), mKate (Shui et al. 2011), eGFP (Nakai, Ohkura, and Imoto 2001), Venus (Nagai et al. 2004), Citrine (Griesbeck et al. 2001), eYFP (Griesbeck et al. 2001).

[0040] In certain embodiments, the circularly permuted fluorescent protein comprises a polypeptide selected from the group consisting of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto.Sequences

[0041] Amino acid residues 147-238 from eGFP: SYNVFIMADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQSKLSKD PNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO:5).

[0042] Amino acid residues 1-146 from eGFP: MVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTT LTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNFN (SEQ ID NO:6).

[0043] cpGFP:SYNVFIMADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQSKLSKD PNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFS VSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEG YIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFN (SEQ ID NO:1).

[0044] hLFABP amino acid residues 1-56: MSFSGKYQLQSQENFEAFMKAIGLPEELIQKGKDIKGVSEIVQNGKHFKFTITAGS (SEQ ID NO:2).

[0045] hLFABP amino acid residues 57-128: KVIQNEFTVGEECELETMTGEKVKTVVQLEGDNKLVTTFKNIKSVTELNGDIITNTMTLGDIVFKR ISKRIL (SEQ ID NO:3).

[0046] Cp.GFP_LFABP: MSFSGKYQLQSQENFEAFMKAIGLPEELIQKGKDIKGVSEIVQNGKHFKFTITAGSGSGSYNVFI MADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKR DHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEG EGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQER TIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNGGKVIQNEFTVGEEC ELETMTGEKVKTVVQLEGDNKLVTTFKNIKSVTELNGDIITNTMTLGDIVFKRISKRIL (SEQ ID NO:4).Method of Use

[0047] The disclosed biosensors can be used to detect per- and polyfluoroalkyl substances (PFAS) in a sample, such as a water sample. In some embodiments, this involves contacting the sample with an effective amount of the disclosed biosensor, and then assaying the sample for fluorescence.

[0048] Fluorescence of the disclosed biosensors can be assayed by any suitable method. For example, fluorescence can be detected by a fluorimeter, a fluorescence microscope, a fluorescence microplate reader, a fluorometric imaging plate reader, or fluorescence-activated cell sorting. In addition, a variety of microscopy techniques known in the art may be used, including, but not limited to, confocal microscopy, two-photon excitation microscopy, light sheet microscopy, or light-field microscopy. See, e.g., FluorescenceMicroscopy: Super-Resolution and other Novel Techniques (A. Cornea, P. M. Conn eds., Academic Press, 2014); E. H. K. Stelzer, K. Greger, and E. G. Reynaud Light Sheet Based Fluorescence Microscopy: Principles and Practice (Wiley- Bl ackwell 1stedition); Handbook of Biological Confocal Microscopy (J. Pawley ed., Springer, 3rdedition, 2006); Light Microscopy: Methods and Protocols (Methods in Molecular Biology, H. Chiarini-Garcia, R. Melo eds., Humana Press, 2011); Confocal and Two-Photon Microscopy: Foundations, Applications and Advances (A. Diaspro ed., Wiley-Liss, 2001); and Cellular Imaging Techniques for Neuroscience and Beyond (F. G. Wouterlood ed., Academic Press, 2012); herein incorporated by reference in their entireties.Production of Biosensors

[0049] The disclosed fluorescent biosensors can be produced in any number of ways, all of which are well known in the art. In one embodiment, the fluorescent biosensors are generated using recombinant techniques. One of skill in the art can readily determine nucleotide sequences that encode the desired polypeptides using standard methodology and the teachings herein. Oligonucleotide probes can be devised based on the known sequences and used to probe genomic or cDNA libraries. The sequences can then be further isolated using standard techniques and, e.g., restriction enzymes employed to truncate the gene at desired portions of the full-length sequence. Similarly, sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce the desired truncations. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA.

[0050] The sequences encoding polypeptides can also be produced synthetically, for example, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311 ; Stemmer et al. (1995) Gene 164:49-53.

[0051] Recombinant techniques are readily used to clone sequences encoding polypeptides useful in the claimed fluorescent biosensors that can then be mutagenized in vitro by the replacement of the appropriate base pair(s) to result in the codon for the desired amino acid. Such a change can include as little as one base pair, effecting a change in a single amino acid, or can encompass several base pair changes. Alternatively, the mutations can be affected using a mismatched primer that hybridizes to the parent nucleotide sequence (generally cDNAcorresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See, e.g., Innis et al, (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci. USA (1982) 79:6409.

[0052] Once coding sequences have been isolated and / or synthesized, they can be cloned into any suitable vector or replicon for expression. As will be apparent from the teachings herein, a wide variety of vectors encoding modified polypeptides can be generated by creating expression constructs which operably link, in various combinations, polynucleotides encoding polypeptides having deletions or mutations therein.

[0053] Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage A (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRI (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pl J61 ( Strep tomyces), pUC6 (Streptomyces) , Ylp5 (Saccharomyces) , YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, generally, DNA Cloning: Vols. I & II, supra; Sambrook et al., supra; B. Perbal, supra.

[0054] Polypeptides can be conveniently synthesized chemically, for example by any of several techniques that are known to those skilled in the peptide art. In general, these methods employ the sequential addition of one or more amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions that allow for the formation of an amide linkage. The protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support, if solid phase synthesis techniques are used) areremoved sequentially or concurrently, to render the final polypeptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce Chemical Co., Rockford, III. 1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis, (Springer- erlag, Berlin 1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, Vol. 1, for classical solution synthesis. These methods are typically used for relatively small polypeptides, i.e. , up to about 50-100 amino acids in length, but are also applicable to larger polypeptides.

[0055] Typical protecting groups include t-butyloxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4- dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o- nitrophenylsulfonyl and the like.

[0056] Typical solid supports are cross-linked polymeric supports. These can include divinylbenzene cross-linked-styrene-based polymers, for example, divinylbenzene- hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and divinylbenzene-benzhydrylaminopolystyrene copolymers.

[0057] Polypeptide analogs can also be chemically prepared by other methods such as by the method of simultaneous multiple peptide synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985) 82:5131-5135; U.S. Pat. No. 4,631,211.

[0058] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1 : A genetically-encoded biosensor for direct detection of perfluorooctanoic acid

[0059] In this study, a biosensing scaffold is introduced that is capable of detecting PFOA in aqueous solutions based on hLFABP and utilizing circularly permuted green fluorescent protein (GFP) that can be further optimized for whole cell detection. This fusionprotein construct shown in Figure 1 , exhibits increased intrinsic fluorescence upon PFOA binding in vitro with a LOD of 235 ppb, a level well within concentration ranges seen in highly contaminated sites. This is also achieved with minimal protein expression and purification steps and no secondary, post purification modifications. The feasibility of this construct to be utilized in vivo through cytosolic E. coli expression is also demonstrated. These results provide a promising detection platform for use in non-aqueous and heterogeneous media detection as whole cell sensors offer robustness, ease of use, application flexibility as compared to sensors based on purified proteins (Gavrilas, S., et al. Sensors. 2022 22:1513).Materials and MethodsMolecular Biology

[0060] For DNA maintenance, E. coli strain DH5a was used and E. coli strain BL21 (DE3) was used for protein expression. Unless otherwise stated, all molecular biology procedures for PCR amplification, plasmid preparation, cell transformation and subcloning were performed according to standard methods supplied by manufacturers. The gene encoding E. coli codon optimized human liver fatty acid binding protein (hLFABP) (NCBI 2168) was previously subcloned into pET-28a(+) using BamHI / Xhol restriction sites (Mann, M. M., et al. Biotech & Bioengineering. 2022 119:513-522) and the cp.GFP.PPYF gene fragment was amplified from EcMBP165-cpGFP.PPYF.pRSET (Addgene plasmid #33372) (Marvin, J. S., et al. Proteins. 2011 79:3025-3036). The cp.GFP.PPYF fragment as well as the destination vector, hLFABP-pET28a(+) were amplified using primer sets FragmentCP.GFP and VectorCP.GFP respectively. The insert and linear vector were then simultaneously digested and ligated via Golden Gate assembly utilizing PaqCI (New England Biolabs). The ligated cpGFP- hLFABP sequence in pET-28a(+) was verified by Sanger Sequencing (Europhins Genomics).FragmentCP.GFP_F: 5' TATCACCTGCACTAggcagcggcagctacaacgtcttcat 3' (SEQ ID NO:11),FragmentCP.GFP_R: 5' TATCACCTGCACTAacccccgttaaagttgtactccagcttg 3' (SEQ ID NO:12), VectorCP.GFP_F: 5'TATCACCTGCACTAgggtaaagtgatccaaaacgaatttaccgttg 3' (SEQ ID NO:13), Vector.CP.GFP_R: 5' TATCACCTGCACTAtgccgctgcccgcggtaat 3' (SEQ ID NO: 14). Protein Expression and Purification

[0061] The recombinant protein was produced after transformation into E. coli BL21 (DE3). Saturated cell solutions were pelleted by centrifugation (3,000 g), resuspended in fresh LB, and grown at 37°C to an QD600 of 0.6. Protein expression was then induced via addition of1mM isopropyl p-D-1-thiogalactopyranoside (IPTG), and cultures transferred to 20°C for 18 hours. Harvested cells were then pelleted by centrifugation (10,000 g) and resuspended in lysis buffer (50mM Tris-CI, 100mM NaCI, 5% v / v glycerol, 1 mM phenylmethylsulfonyl fluoride) before lysis via sonication. The clarified supernatant was then purified by Ni2+-affinity chromatography using Chelating Sepharose Fast Flow (Cytiva). The 1 mL column was equilibrated with 50 mM Tris-CI buffer (pH 8) containing 10 mM imidazole, and protein was separated using a stepwise elution of imidazole up to 500 mM. The fractions collected were analyzed by SDS-PAGE to determine purity. Pure fractions were then pooled and dialyzed against phosphate buffered saline (PBS) pH 7.6. Concentration of all protein samples was measured using a Pierce BCA protein assay kit.Spectroscopy

[0062] All absorbance and fluorescence measurements for cpGFP.hLFABP were performed using a Synergy Neo2 Hybrid Multi-Mode Microplate Reader (Biotek) at room temperature and under steady state conditions.In vitro assays

[0063] PFOA binding assays in vitro were performed by titrating PFOA into protein (1 pM final concentration) in either PBS buffer (pH 7.6) or creek water taken from the Dell and Meadow Creek on the grounds of University of Virginia. To determine sensor ability in systems containing other anionic surfactants, the assay was also performed in PBS buffer (pH 7.5) with sodium dodecyl sulfate (SDS) at a final concentration of 1 pM. Samples were allowed to equilibrate for 5 minutes before fluorescence spectra were recorded over 500-600 nm after excitation at 395 and 485 nm. For more quantitative data, fluorescence intensity endpoint reads at 510 nm after excitation at 485 nm were also collected. The dissociation constant (Kd) was determined by fitting the fractional change in fluorescence intensity (F) at 510nm and corresponding PFOA concentrations to a one-site binding model using nonlinear regression after correcting for protein in buffer only fluorescence:In vivo assays

[0064] For in vivo assays, the induced cells were harvested and resuspended in PBS (pH 7.6) to cut down on background media fluorescence. After titration with PFOA, cells were allowed to equilibrate for 30 minutes at room temperature. After 30 seconds of orbital shaking, the fluorescence spectra as well as endpoint data were collected as described above. After fluorescence data was normalized by OD600, the fractional change in fluorescence intensity at510nm was plotted against PFOA concentration. However, to account for more nonspecific interactions, the data were fit to a log-dose response model where a half maximal effective concentration (EC50) was obtained:Results and Discussion

[0065] Fluorescent proteins (FPs) contain optical properties that are extremely dependent on the microenvironment surrounding their chromophores (Oltrogge, L. M., et al. Biochemistry. 53:5947-5957; Tsien, R. Y. Annu. Rev. Biochem. 1998 67:509-544). This chromophore sensitivity has led to the widespread use of FPs like GFP as sensing tools since small changes to protonation equilibrium are transduced easily. This can be through direct interaction of analytes and chromophores like with FP based pH and ion sensors (Hess, S. T., et al. J. Phys. Chem. B. 108:10138-10148) or through the addition of a separate binding unit where ligand induced conformation change leads FRET-based activity or even allosteric based fluorescence changes (Doi, N., et al. FEBS Letters. 1999 453:305-307; Hires, S. A., et al. Brain Cell Bio. 2008 36:69-86). While the addition of ligand-binding receptors to FPs is conducive for binding events that lead to large changes in receptor conformation, the use of circularly permuted GFP has been proven to be capable of transducing binding events for proteins with a wide range of conformational flexibilities (Sakaguchi, R., et al. Bioorganic & Medicinal Chemistry. 2009 17:7381-7386). The circular permutation process involves fusing the natural GFP termini which forms new termini that can then be fused to insert a receptor of interest closer to the chromophore (Baird, G. S., et al. Proc. Natl. Acad. Sci. U.S.A. 1999 96:11241- 11246).

[0066] To create a sensor capable of detecting PFOA, circularly permuted GFP (cpGFP) and human liver fatty acid binding protein were utilized as optical signal and recognition units respectively. The chosen receptor, hLFABP has not only been shown to bind PFOA with moderate affinity (Cheng, W., et al. Environ. Sci. Technol. 2018 52:7972-7980; Sheng, N., et al. Arch Toxicol. 2016 90:217-227) but has also been used previously as a scaffold for a PFAS biosensor not suitable for genetic encoding (Mann, M. M., et al. Biotech & Bioengineering. 2022 119:513-522). However, hLFABP is not known to have extensive conformational changes upon binding of endogenous fatty acids (Cai, J., et al. Biophysical Journal. 2012 102:2585-2594; Sharma, A., et al. J. Biol. Chem. 2011 286:31924-31928), and the little structural information of PFOA binding shows only minimal changes in alpha helical composition (Zhang, L., et al.Environ. Sci. Technol. 2013 47:11293-11301 ; Sheng, N„ et al. Arch Toxicol. 2016 90:217-227). Therefore, it is imperative that the cpGFP construct is fused near the PFOA binding region of hLFABP without interrupting residues directly responsible for ligand interactions. The residues S56 and K57 in the loop region shown in Figure 1 were chosen for domain splitting. While located in a flexible region away from residues taking part in electrostatic interactions (Cheng, W., et al. Environ. Sci. Technol. 2018 52:7972-7980; Sheng, N., et al. Arch Toxicol. 2016 90:217-227), S56 and K57 are in a region where modest change in structure occurs upon binding of palmitic acid as shown in Figure 1 as the overlay of the apo and holo form of hLFABP. (PDB 3STN and 3STK respectively) (Sharma, A., et al. J. Biol. Chem. 2011 286:31924-31928). Furthermore, previous work has shown insertion of the fluorophore, acrylodan, into a reasonably close residue (F50) of hLFABP is able to probe PFOA binding (Mann, M. M., et al. Biotech & Bioengineering. 2022 119:513-522). Figure 1 also shows a linear map of the construct as well as a graphical overview of the sensor function. As ligand is bound by the split hLFABP, the change in microenvironment around cpGFP’s chromophore will elicit a change in fluorescence. Specifically, this change is seen as an increase in fluorescence after excitation.

[0067] The designed construct was subsequently cloned as described above, expressed in E. coli BL21 (DE3), and purified via immobilized metal affinity chromatography. Purified fractions containing the single band of cpGFP. hLFABP at -46.5 kDa (Figure 2) were then dialyzed and used for in vitro analysis.

[0068] Enhanced green fluorescent protein (EGFP), the original basis of this cpGFP signal unit, as well as cpGFP. hLFABP fusion have absorbance peaks (Figure 3A) corresponding with the protonated and deprotonated forms at ~395nm and ~490nm respectively (Cormack, B. P., et al. Gene. 1996 173:33-38). Upon titration of PFOA, the absorbance at 395nm increased and decreased at 485nm. This indicates shifts in the equilibrium state of the chromophore from protonated to deprotonated states upon binding of PFOA to the split hLFABP.

[0069] Fluorescence response at these two wavelengths was assessed upon addition of PFOA. Figure 3B & 3C show that while emission spectra changes occur at both wavelengths, excitation at 485nm exhibits a much more exaggerated response in overall fluorescence intensity change.

[0070] Therefore, to quantify binding of PFOA to cpGFP. hLFABP, endpoint intensity at 510nm after excitation at 485nm was used. Figure 4 shows binding of PFOA to cpGFP. hLFABP in PBS (pH 7.6) as a fractional change in fluorescence. This data is shown fitted to a one sitebinding model based on the best fit to experimental data. This is consistent with studies suggesting PFOA binds in a 1 :1 stoichiometry to WT hLFABP despite cpGFP.hLFABP utilizing a split hLFABP domain (Zhang, L., et al. Environ. Sci. Technol. 201347:11293-11301 ; Mann, M. M., et al. Biotech & Bioengineering. 2022 119:513-522; Sheng, N., et al. Arch Toxicol. 2016 90:217-227). The calculated dissociation constant Kd was determined to be 11.9 ± 1.6 iM which is also consistent with previous studies characterizing binding of PFOA to WT hLFABP and variants (Zhang, L., et al. Environ. Sci. Technol. 201347:11293-11301 ; Mann, M. M., et al. Biotech & Bioengineering. 2022 119:513-522; Sheng, N., et al. Arch Toxicol. 2016 90:217-227; Sheng, N., et al. Arch Toxicol. 2018 92:359-369).

[0071] To assess feasibility and selectivity of the sensor in more realistic application systems, these experiments were also done with spiked water samples taken from the Dell and Meadow Creek on the grounds of University of Virginia as well as in buffer containing the anionic surfactant, sodium dodecyl sulfate (SDS), as a competitor for binding (Figure 4). While Kdvalues obtained in all buffer systems were similar as shown in Figure 4, the maximum response, and limits of detection (LCDs) varied. While maximum response, (AF / F0)max, is reduced due to outside allosteric interactions in creek water-based assays, the overall binding affinity and limits of detection remain comparable to PBS based data with LCDs in the hundreds of ppb. While this limit is pushed toward the part per million for buffer containing SDS, the ability to detect PFOA at this level in a system with a known hLFABP binder is promising for application of samples that contain other anionic surfactant co-contaminants.In vivo

[0072] One of the most valuable aspects of genetically encoded sensors is the ability to be used in vivo. Specifically, in environmental detection, utilizing whole bacterial cells introduces a robustness to a sensor as compared to proteins alone in terms of tolerance to physical changes like pH and temperature as cell membranes act as a barrier from harsh environmental conditions (Koebnik, R., et al. Mol Microbiol. 2000 37:239-253). Furthermore, whole cell-based systems are usually more amenable to immobilization-based implementation than proteins (Su, L., et al. Biosensors and Bioelectronics. 2011 26:1788-1799) and offer advantages such as portability and the possibility of detection in complex matrices with minimal sample preparation (Lei, Y., et al. Analytica Chimica Acta. 2006 568:200-210).

[0073] Therefore, as a first pass at feasibility of whole cell detection, E. coli cells expressing cpGFP.hLFABP cytosolically were incubated with PFOA, and changes in fluorescence monitored. The increase in fluorescence upon PFOA titration is shown in Figure 5. To account for complications the bacterial interactions bring, the data was fit to a log-doseresponse model rather than a one site binding model. While only eliciting a maximal response of around 10%, the sensor expressing cells are capable of detecting PFOA in PBS (pH 7.6) with a LOD of 7.1 ±1.8 ppm.

[0074] This study demonstrates the design and initial applicability of an intrinsic fluorescent PFOA sensor based on conjugation of cpGFP to a split hLFABP construct. The purified fusion protein exhibited dose-dependent changes in absorption spectra as well as fluorescence intensity that demonstrate saturation behavior. This shows that despite small changes in structural conformation, splitting hLFABP allows PFOA induced changes large enough for transduction by cpGFP without detrimental changes to binding as compared to wild type hLFABP.

[0075] Direct detection of PFOA was achieved in buffer as well spiked water samples with LODs within relative environmental concentration ranges without sample pre-treatment. The feasibility of utilizing this construct was also Demonstrate in whole bacterial cells through cytosolic expression to detect PFOA. This is extremely promising as minimal optimization was done in terms of individual GFP and hLFABP moieties. As has been shown prior, just optimizing the linkers between cpGFP and receptor proteins can improve optical signal by more than 200% (Marvin, J. S., et al. Proteins. 2011 79:3025-3036).

[0076] As further evidence continues to elucidate the wide-reaching contamination of PFAS chemicals, the need for detection strategies grows. Specifically, detection in a wide variety of sample types and concentrations are necessary. As mentioned above, biosolids and irrigation waters have become reservoirs as well as transporters of PFAS to crops, livestock, and subsequently, people. In the last 10 years, PFAS have been found at detectable levels not only in food crops but also livestock, including milk from dairies with groundwater and hay contamination (Death, C., et al. Science of The Total Environment. 2021 774:144795). Therefore, to address the need for easy detection in all sample types, our lab looks towards utilizing synthetic biology. At the bench scale, biosensor feasibility studies are usually conducting in liquid culture. However, genetically encoded biosensors have the capability for a multitude of applications including immobilization for the design of test strips, and microfluidic devices (Gavrilas, S., et al. Sensors. 2022 22:1513; Su, L, et al. Biosensors and Bioelectronics. 2011 26:1788-1799). With biological tool kits, researchers have the capability to modify proteins and enzymes to enhance sensitivity and selectivity drastically. Furthermore, genetically encoded biosensors can be designed with multiple functions. By introducing new proteins and enzymes or creating genetic circuits, whole cell sensors seem to have unlimited capabilities, including pre-treatment simplification. Therefore, by demonstrating cpGFP. hLFABP as a promisingplatform for intrinsic fluorescent-based detection, we hope to enable further synthetic biologybased approaches for PFAS detection.

[0077] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0078] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. A biosensor fusion protein comprising an N-terminal half of a human liver fatty acid binding protein (hLFABP) and a C-terminal half of the hLFABP separated by a circularly permuted fluorescent protein (cpFP), wherein the binding of the N-terminal half of the hLFABP and the C-terminal half of the of the hLFABP to a per- or polyfluoroalkyl substance (PFAS) elicits a change in fluorescence of the cpFP.

2. The biosensor fusion protein of claim 1 , wherein the N-terminal half of hLFABP comprises amino acids 1-56 of the hLFABP, and wherein the C-terminal half of hLFABP comprises amino acids 57-128 of the hLFABP.

3. The biosensor fusion protein of claim 1 or 2, wherein the fusion protein comprises the formula:N-hLFABP -1- FP-C -2- N-FP -3- hLFABP-C wherein “N-hLFABP” represents the N-terminal half of the hLFABP; wherein “hLFABP-C” represents the C-terminal half of the hLFABP; wherein “FP-C” represents an N-terminal half of the cpFP; wherein “N-FP” represents a C-terminal half of the cpFP; and wherein“-2-”, and “-3-” represents a first, second, and third flexible linker, respectively.

4. The biosensor fusion protein of any one of claims 1 to 3, wherein the cpFP is a circularly permuted green fluorescent protein (cpGFP)5. The biosensor fusion protein of claim 4, wherein the cpGFP comprises amino acids 147- 238 of GFP linked to amino acids 1-146 of GFP by the second flexible linker.

6. The biosensor fusion protein of claim 5, wherein the second flexible linker comprises the amino acid sequence GGTGGS (SEQ ID NO:7), GGSGGS (SEQ ID NO:8), or GGTGGSGGTGGS (SEQ ID NO:9).

7. The biosensor fusion protein of claim 6, wherein the cpGFP comprises the amino acid sequence SEQ ID NO:1.

8. The biosensor fusion protein of any one of claims 1 to 7, wherein the first flexible linker comprises the amino acid sequence GG, GSG, or GGSGGS (SEQ ID NO:8).

9. The biosensor fusion protein of any one of claims 1 to 8, wherein the third flexible linker comprises the amino acid sequence GG, GSG, or GGSGGS (SEQ ID NO:8).

10. The biosensor fusion protein of any one of claims 1 to 9, wherein the N-terminal half of hLFABP comprises the amino acid sequence SEQ ID NO:2, and wherein the C-terminal half of hLFABP comprises the amino acid sequence SEQ ID NO:3.

11. The biosensor fusion protein of claim 1 , comprising the amino acid sequence SEQ I D NO:4.

12. A cell engineered to express the biosensor fusion protein of any of claims 1 to 11 .

13. The cell of claim 12, wherein the cell is a strain of E. coli with a membrane permeable to per- and polyfluoroalkyl substances (PFAS).

14. The cell of claim 13, wherein the cell is an AS19 or BL21 strain of E. coli.

15. A method for detecting per- and polyfluoroalkyl substances (PFAS) in a sample, comprising contacting the sample with the biosensor of any one of claims 1 to 11 or the cell of any one of claims 12 to 14, assaying the sample for fluorescence, wherein an increase in fluorescence is an indication of the presence of PFAS.

16. The method of claim 15, wherein the PFAS is perfluorooctanoic acid (PFOA).

17. The method of claim 15 or 16, wherein the sample is a water sample.

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