Multiplexed nanomechanical cantilevers for per- and polyfluoroalkyl substance detection

Abstract

Disclosed herein is a system for detecting a per- and polyfluoroalkyl substance (PFAS), including a first microcantilever; and one or more polypeptides coupled to at least a portion of the first microcantilever, wherein interaction of the one or more polypeptides with the per- and polyfluoroalkyl substance (PFAS) induces a detectable deflection in the first microcantilever. Further disclosed is a method for detecting a per- and polyfluoroalkyl substance (PFAS), including exposing a sample containing a per- and polyfluoroalkyl substance (PFAS) to a first polypeptide functionalized microcantilever including: a first microcantilever, and one or more polypeptides coupled to at least a portion of the first microcantilever, wherein interaction of the one or more polypeptides with the per- and polyfluoroalkyl substance (PFAS) induces a detectable deflection in the first microcantilever; and detecting a movement of the microcantilever.

Description

[0001] MULTIPLEXED NANOMECHANICAL CANTILEVERS FOR PER- AND POLYFLUOROALKYL SUBSTANCE DETECTION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority to U.S. Provisional Patent Application No. 63 / 735,427 that was filed December 18, 2024. The entire contents of which are hereby incorporated by reference.

[0004] FIELD OF THE INVENTION

[0005] The disclosed technology is generally directed to detection of per- and polyfluoroalkyl substances (PFAS). More particularly the technology is directed to microcantilever detection of PFAS.

[0006] BACKGROUND OF THE INVENTION

[0007] Synthetic chemicals called per- and polyfluoroalkyl substances (PFAS) are characterized by the replacement of each hydrogen atom on an alkyl chain with a fluorine atom. These chemicals consist of a hydrophobic carbon-fluorine (C-F) chain and a hydrophilic functional group (carboxyl for PFOA and sulfonate for PFOS). The carbon-fluorine bond is exceptionally stable due to its high bond dissociation energy, making these compounds highly resistant to degradation that are widespread in the environment and pose significant risks to human health. These substances have been extensively utilized in numerous industrial applications and consumer products due to their unique surface tension lowering and surface wettability.

[0008] PFOA and PFOS are commonly known as "forever chemicals" because of their remarkable resistance to environmental degradation. These substances can remain in the environment for many years and have been found in water, soil, air, and even in the blood of humans and wildlife worldwide. Their extensive presence is due to both direct emissions from industrial facilities and the breakdown of consumer products containing these chemicals. Further, extensive research has identified various health concerns related to PFOA and PFOS exposure, including carcinogenicity, reproductive and developmental toxicity, endocrine disruption, liver damage, kidney disease, and gastrointestinal disorders.

[0009] 1

[0010] QB\702581.02730\100020683.1 Detecting PFOS (Perfluorooctane Sulfonic Acid) and PFOA (Perfluorooctanoic Acid) in environmental and biological samples is crucial due to their widespread presence and potential health risks. Ongoing improvements in detection methods and regulatory standards are crucial to addressing the challenges posed by PFOS and PFOA contamination.

[0011] BRIEF SUMMARY OF THE INVENTION

[0012] Disclosed herein are systems and methods for detecting a per- and polyfluoroalkyl substance (PFAS) using nanomechanical sensors, such as microcantilevers. These systems and methods enable very low limits of detection for PFAS molecules and achieving the highest sensitivity for PFAS reported. The system for detecting a PFAS may comprise a microcantilever; and one or more polypeptides coupled to at least a portion of the microcantilever, wherein interaction of the one or more polypeptides with the PFAS induces a detectable deflection in the first microcantilever. Suitably, contact of a sample comprising less than 100 ppm, less than 10 ppm, less than 1 ppm, less than 100 ppt, less than 10 ppt, or about 1 ppt of the PFAS with the one or more polypeptides induces a detectable deflection in the microcantilever.

[0013] Another aspect of the invention provides for a method for detecting a PFAS. The method may comprise exposing a sample containing a PFAS to a polypeptide functionalized microcantilever and detecting a movement of the polypeptide functionalized microcantilever. The polypeptide functionalized microcantilever comprises a microcantilever and one or more polypeptides coupled to at least a portion of the first microcantilever, wherein interaction of the one or more polypeptides with the PFAS induces a detectable deflection in the first microcantilever.

[0014] Also disclosed herein are detectable complexes. The detectable complexes include a polypeptide, having a binding site and an exposed amino acid residue, and a per- and polyfluoroalkyl substance (PFAS). The PFAS has a fluoroalkyl tail and a polar terminal group within the binding site. The detectable complexes include a polar terminal group interacting with the exposed amino acid residues.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically

[0017] 2

[0018] QB\702581.02730\100020683.1 represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0019] Figure 1 shows, in panel a), concentration-dependent response curves for HFBA detection on L-FABP antibody (20pg / mL, respectively) functionalized microcantilevers. Panel b) shows concentration-dependent response curves for PFOA / PFSA detection on L-FABP antibody (20pg / mL, respectively) functionalized microcantilevers.

[0020] Figure 2 shows, in panel a), concentration-dependent response curves for HFBA detection on anti-albumin antibody (20pg / mL, respectively) functionalized microcantilevers. Panel b) shows concentration-dependent response curves for PFOA / PFSA detection on anti-albumin antibody (2pg / mL, respectively) functionalized microcantilevers.

[0021] Figure 3 shows an exemplary system of some embodiments disclosed herein. Dotted lines show optional aspects.

[0022] Figure 4 shows an exemplary method of some embodiments disclosed herein. Dotted lines show optional aspects.

[0023] Figure 5 shows dose-dependent detection of PFA derivatives using anti-albumin antibody immobilized microcantilever platform. Panel (a) shows HFBA, panel (b) shows PFOA, panel (c) shows PFOS, and panel (d) shows PFOSA. Deflection signals indicate specific binding interactions at varying concentrations. Dose-dependent response curves for the detection of PFA derivatives at the indicated concentrations in DI Water. The curves represent deflection of the microcantilever functionalized with an anti-albumin antibody (2pg / mL) as measured by f- AFM over the course of 12 minutes. Graphs show the average of triplicate deflection measurements in nm + / - standard deviation per timepoint and condition.

[0024] Figure 6 shows dose-dependent detection of PFA derivatives using anti-albumin antibody immobilized microcantilever platform: Panel (a) shows HFBA, panel (b) shows PFOA, panel (c) shows PFOS, and panel (d) shows PFOSA. Deflection signals indicate specific binding interactions at varying concentrations. Dose-dependent response curves for the detection of PFA derivatives at the indicated concentrations in DI Water. The curves represent deflection of the microcantilever functionalized with an anti-albumin antibody (2pg / mL) as measured by f- AFM over the course of 12 minutes. Graphs show the average of triplicate deflection measurements in nm + / - standard deviation per timepoint and condition.

[0025] 3

[0026] QB\702581.02730\100020683.1 Figure 7 shows a system for nanomechanical detection of PFAS, as disclosed in some examples herein. Specifically, the system shows an optical detection scheme of PFAS detection using a polypeptide-functionalized microcantilever surface.

[0027] Figure 8 shows a schematic depicting an active microcantilever with bound polypeptides which, upon interacting with a PFAS analyte, form a detectable complex. Also shown is a reference microcantilever. Each microcantilever is shown with integrated metal oxide semiconductor field effect transistor (MOSFET) for electrical differential readout of microcantilever deflection.

[0028] Figure 9 shows a schematic of one example of microcantilever functionalization as disclosed herein. The schematic shows the functionalization of a gold microcantilever surface with a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MU A), followed by EDC- NHS chemistry to further functionalize the SAM with polypeptides, thereby immbolizing the polypeptides on the microcantilever surface.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] Disclosed herein are systems and methods for detecting a per- and polyfluoroalkyl substance (PFAS) using nanomechanical sensors, such as microcantilevers. These systems and methods enable very low limits of detection for PFAS molecules and achieving the highest sensitivity for PFAS reported.

[0031] The systems and methods disclosed herein include a first microcantilever and one or more polypeptides coupled to at least a portion of the first microcantilever. The interaction of the one or more polypeptides with a per- and polyfluoroalkyl substance (PFAS) induces a detectable deflection in the first microcantilever.

[0032] As used herein, the term ‘PFAS’ refers to free acid forms, salt forms, or other chemical states as applicable to a particular sample of per- and polyfluoroalkyl substance. In some embodiments, PFOS refers to perfluorooctanesulfonic acid (free acid), and PFOS potassium salt refers to a salt form. In some embodiments, PFSA refers to perfluoroalkyl sulfonates and may include PFOS and / or salts thereof. In some embodiments, PFCA refers to perfluoroalkyl carboxylates and may include PFOA and / or salts thereof

[0033] PFAS may include a fluoroalkyl tail. The fluoroalkyl tail may include fluoroalkyl tails including C1-C20. The fluoroalkyl tail may be fully fluorinated or partially fluorinated. In some cases, the fluoroalkyl tail may include H, Cl, Br, or I. In some embodiments, the fluoroalkyl tail 4

[0034] QB\702581.02730\100020683.1 may include perfluorobutyl (C4F9), as in the fluoroalkyl tail of perfluorobutanesulfonic acid (PFBS) and perfluorobutanoic acid (PFBA), perfluorohexyl (-C6F13) as in the fluoroalkyl tail of perfluorohexanesulfonic acid (PFHxS) and perfluorohexanoic acid (PFHxA), perfluoroheptyl (- C7F15) as in the fluoroalkyl tail of perfluoroheptanoic acid (PFHpA), perfluorooctyl (-CgFn) as in the fluoroalkyl tail of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), perfluorononyl (-C9F19) as in the fluoroalkyl tail of perfluorononanoic acid (PFNA), or Perfluorodecyl (-C10F21) as in the fluoroalkyl tail of perfluorodecanoic acid (PFDA).

[0035] PFAS further include a polar terminal group. In some cases, the further functional groups may include carboxylic acids (-COOH or carboxylates -COO-), sulfonic acids (-SO3H or sulfonates -SO3 ), phosphonic and phosphinic acids (phosphonates and phosphinates), alcohols, sulfonamides, ethers, amides, or any combinations thereof.

[0036] Per- and polyfluoroalkyl substances may include perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates (PFSAs). Examples of PFAS include perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), perfluoroocatanesulfonate (PFOS), perfluorooctanesulfonamide (PFOSA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorohexane sulfonic acid (PFHxS), and salts thereof. In some cases, the per- and polyfluoroalkyl substances (PFAS) is a perfluoroalkyl carboxylic acid.

[0037] The system and methods disclosed herein may include a microcantilever and a detector for detecting a response of the microcantilever to the sample and generating a signal indicative of the response of the microcantilever to the sample. Suitably, the detector is configured to detect a response such as a bending moment, also known as deflection. In some cases, the deflection includes surface stress deflection which refers to static bending of a cantilever due to differential surface stress caused by analyte binding. In some cases, the differential surface stress is compressive. An example of a suitable system and method includes an atomic force microscopy measurement mode used to monitor cantilever deflection as a function of time during exposure to a sample (sometimes abbreviated as ‘f-AFM’). In some embodiments, f-AFM refers to a functional AFM measurement protocol configured to provide real-time cantilever deflection output during analyte exposure.

[0038] The systems and methods described herein may include two or more microcantilevers (e.g., a first cantilever, a second cantilever, etc), thereby allowing for multiplexed detection. Each

[0039] 5

[0040] QB\702581.02730\100020683.1 microcantilever may be functionalized with different polypeptides useful in detecting the presence of one or more PFAS molecules. The systems and methods disclosed herein may further include a control or reference microcantilever. In some cases, the control or reference cantilever may be used to perform differential measurements as compared to one or more polypeptide functionalized microcantilevers, a seen in FIG. 8. The control or reference microcantilever may be functionalized substantially identically to a polypeptide functionalized microcantilever and be further treated to prevent PFAS molecule interactions, or the control or reference microcantilever may not be functionalized. The control or reference microcantilever may include gold, silicon nitride, or other materials.

[0041] One aspect of the invention provides for a nanomechanical sensor comprising a polypeptide functionalized microcantilever. As used herein, "polypeptide functionalized" or “polypeptide coupled” means a microcantilever including a tethered polypeptide where interaction of a PFAS molecule in a sample to the tethered polypeptide results in a detectable deflection of the microcantilever. The Examples demonstrate the use of polypeptide functionalized microcantilevers where a polypeptide is tethered to the microcantilever and a response is generated by binding of a PFAS molecule in a sample to the tethered polypeptide or binding a PFAS-bound complex in a sample to the tethered polypeptide. The reverse, where a PFAS molecule is tethered to the microcantilever and a polypeptide is present in a sample, may also generate a detectable deflection of the microcantilever by interacting with the tethered PFAS molecule.

[0042] Polypeptide functionalized microcantilevers comprise a microcantilever, a polypeptide configured to interact with a PFAS molecule, and a tether covalently tethering the polypeptide to the microcantilever. PFAS functionalized microcantilevers comprise a microcantilever, a PFAS molecule configured to interact with a polypeptide molecule, and a tether covalently tethering the PFAS molecule to the microcantilever.

[0043] As used herein, the term “polypeptide” refers to a linear organic polymer of amino acids and complexes thereof. Polypeptides may include any number of amino acids. Suitably, the polypeptide may be a short-chain polypeptide, having from about 4 to about 50 amino acids, or the polypeptide may be a long-chain polypeptide, having more than about 50 amino acids. Polypeptides may include single full-length proteins or fragments thereof. Examples of polypeptides include antibodies (e.g., IgA, IgD, IgE, IgG, or IgM), minibinder, nanobodies, an antibody fragment (e g., Fab or F(ab’)2), single-domain antibody (e.g., sdAb), or an engineered or

[0044] 6

[0045] QB\702581.02730\100020683.1 recombinant antibody or fragment thereof (e.g., scFv, di-scFV, or tri-scFV). In some examples, polypeptides include one or more antibodies. Antibodies may refer to any immunoglobulin, including mono / polyclonal, fragments, or engineered binders, with affinity for a PFAS binding protein epitope or PFAS mimetic motif. In some examples, antibodies include anti L FABP, anti HAS, anti albumin, and any combinations thereof. The Examples demonstrate Liver-type fatty acid-binding protein (L-FABP) and antialbumin antibodies may be utilized, however other polypeptides may be used. Nanobodies or minibinder proteins offer improved characteristics over traditional monoclonal antibodies for diagnostics. Because nanobodies or minibinders are significantly smaller than monoclonal antibodies, e.g., about 15 kDa as compared to their 150 kDa antibody counterparts, their theoretical packing efficiency increases, allowing more binders to be immobilized and theoretically more antigens to be immobilized and detected.

[0046] In some embodiments, the one or more polypeptides coupled to a microcantilever includes antibodies directed against liver-type fatty acid-binding protein (L-FABP). Anti-L-FABP antibodies provide broad PFAS recognition due to hydrophobic and amphipathic binding pockets that interact with perfluoroalkyl chains of varying length and terminal chemistry. In some embodiments, anti-L-FABP functionalized microcantilevers exhibit strong and concentrationdependent deflection responses across multiple PFAS species, including perfluoroalkyl carboxylates and perfluoroalkyl sulfonates.

[0047] In some embodiments, the one or more polypeptides coupled to a microcantilever include anti-albumin antibodies. Anti-albumin antibodies enable detection of PFAS that associate with albumin in biological and environmental matrices and may exhibit different selectivity and sensitivity profiles relative to anti-L-FABP antibodies. As shown in the Examples,, anti-albumin functionalized microcantilevers preferentially respond to specific PFAS chemotypes or concentration ranges. The use of distinct antibodies provides complementary detection modalities within the disclosed system.

[0048] The polypeptide functionalized microcantilevers comprise an effective surface density of tethered polypeptides. As used herein, "effective surface density" means an amount of tethered polypeptides that when in contact with sample comprising one or more PFAS molecules is capable of inducing a detectable response. Polypeptide functionalized microcantilevers may be prepared by contacting tether-functionalized microcantilevers with a precursor solution comprising polypeptides for the desired target such that the polypeptides can covalently bond to the tethers. In

[0049] 7

[0050] QB\702581.02730\100020683.1 some embodiments, the precursor solution comprises between 0.1 pg / ml - 100 pg / ml of the polypeptide to be bound.

[0051] In some embodiments, the effective surface density is capable of inducing a detectable response when the sample comprises at least 100 ppm, 10 ppm, 1.0 ppm, 0.01 ppm, 100 ppt, 10 ppt, and 1.0 ppt of aPFAS molecule. In some embodiments, the effective surface density is capable of inducing a detectable response when the sample contains about 1 ppt of a PFAS molecule. The sample containing a PFAS molecule may be a solid, liquid, or gas.

[0052] The microcantilever is composed of one or more materials that can respond to polypeptide- PFAS molecule interactions and covalently bond with a tether. Suitably, the microcantilever may be composed of silicon coated with gold as in the Examples but other materials may also be used.

[0053] The microcantilever is also dimensioned to allow for a response in response to polypeptide- PFAS molecule interaction (i.e., the formation of a detectable complex including a polypeptide and one or more PFAS). The microcantilever may have a maximum dimension of 100 pm or more, 1.5 mm or less, or of from about 100 pm to about 600 pm. The microcantilever may also be dimensioned to have length from about 250 pm to 600 pm, and width of 50 pm tolOO pm with thickness of 1 pm to 2 pm. The cantilevers may be tipped or tipless.

[0054] The tethers covalently couple the polypeptide to the microcantilever. The tether may be a heterobifunctional molecule with a spacer between immobilization and conjugation functional moieties. The Examples demonstrate the use of a thiocarboxylic acid, where the thiol group is used to immobilize the tether to the microcantilever via gold-thiol bonding, the carboxylic acid moiety is used to conjugate the antibody to the tether, and an alkyl chain provides a spacing and conformation flexibility between the polypeptide and microcantilever. The immobilization group, conjugation group, and spacer may be appropriately selected based on the material of the microcantilever and polypeptide to be conjugated. As shown in FIG. 9, in some cases, a selfassembled monolayer (SAM) may be used as the immobilization group and / or the spacer group. In some cases, a SAM may be formed on a gold surface of a microcantilever. In some cases, the SAM may include 11-mercaptoundecanoic acid (MU A). Optionally, a spacer may be disposed between the immobilization group and the polypeptide. In some cases, the spacer may include an amine. In one example, 3-aminophenylboronic acid (APBA) may be the spacer. Conjugation is a chemical strategy for forming stable covalent linked between molecules, where one is a biomolecule such as the antibodies and antigens used herein. Conjugation chemistries often use

[0055] 8

[0056] QB\702581.02730\100020683.1 carboxyl, amine, thiol, or bio-orthogonal groups that target functional groups that are not native in biomolecules. The Examples demonstrate the use of l-ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling (EDC-NHS chemistry), but other conjugation chemistry may also be used and are generally known in the art.

[0057] In some cases, the functionalized microcantilever further comprises a surface area substantially-free of polypeptides. The surface area substantially-free of polypeptides may be coupled to bovine serum albumin. In some cases, the bovine serum albumin may prevent nonspecific PFAS molecule interactions with the microcantilever.

[0058] In some embodiments, the detector can detect a bending moment response. In some embodiments, the bending moment is detected by optically detecting a bending moment. In other embodiments, the bending moment is detected by electrically detecting a bending moment. In some embodiments, the system is configured for multiplexed detection. Suitably, the system may include two or more polypeptide functionalized cantilevers where different polypeptides are tethered to different cantilevers. Where the system comprises two or more polypeptide functionalize cantilevers, the detector may be configured to individually detect the response of each cantilever and generate a signal indicative of the response of each sensor. Multiplexed arrays enable simultaneous detection of multiple PFAS species, comparative analysis across different binders, and improved analytical robustness. The system may further include one or more reference microcantilevers. Reference microcantilevers may be non-functionalized or functionalized substantially identically to a polypeptide functionalized microcantilever and further treated to prevent PFAS molecule interactions. For example, the polypeptide may be treated with heat, high salinity, or chemical agents such as formaldehyde or a formaldehyde derivative to prevent or change the interaction of the treated polypeptide with PFAS. Reference microcantilevers enable differential measurements that compensate for thermal drift, fluidic- induced stress, nonspecific adsorption, and environmental perturbations. Differential subtraction of reference microcantilever response from polypeptide functionalized microcantilever response improves selectivity, sensitivity, and measurement stability.

[0059] The systems and methods detect deflection in a microcantilever by atomic force microscopy methods, such as optical detection (as shown in FIG. 7), or electrical detection methods (as shown in FIG. 9). Electrical detection of the deflection of the microcantilever may be

[0060] 9

[0061] QB\702581.02730\100020683.1 accomplished by a with an integrated field effect transistor (FET), such as a MOSFET (Metal Oxide Semiconductor Field Effect Transistor) transistor. In some cases, the MOSFET is further combined with a bipolar transistor, and the combined circuit of MOSFET and bipolar transistors is called BiMOS. The MOSFET and / or BiMOS is placed at one or more high stress regions of the microcantilever and functions as a transducer of microcantilever stress (e.g., tensile, or compressive stress) as a product of polypeptide-PFAS molecule interaction, into a measurable electrical signal. Suitable devices, systems, and methods of using for electrical detection of deflection of microcantilevers are described in U.S. Patent No. 7,157,897, U.S. Patent No. 7,759,924, and U.S. Patent Application No. 2024 / 0277252 each of which are incorporated by reference, in their entireties, herein.

[0062] In some embodiments, microcantilever deflection is detected optically by reflecting light from the microcantilever onto a position sensitive detector. Optical detection enables high- resolution measurement of surface-stress-induced deflection and is compatible with laboratory and benchtop instrumentation.

[0063] In some embodiments, microcantilever deflection is detected electrically using integrated transducers, including metal oxide semiconductor field effect transistors (MOSFETs), bipolar- MOS (BiMOS) devices, piezoresistive elements, or combinations thereof. Electrical detection converts mechanical stress or strain into an electrical signal and enables compact, integrated, and portable system implementations. Optical and electrical readout modalities may be used independently or in combination.

[0064] Optical detection of the deflection of a microcantilever has been described in detail elsewhere in the art. Briefly, optical detection may be accomplished by reflecting laser light off of at least a portion of the microcantilever and detecting the reflected laser light on a position sensitive detector, such as a quadrant photodiode. When the microcantilever is undeflected, half of the reflected laser spot reaches the top quadrants of the photodiode, and the other half the bottom quadrants. The difference between the photovoltage output by the top (KP) and the bottom quadrants ( ott™) is zero (AK= K»P>Kott™). When the microcantilever interacts with the a per- and polyfluoroalkyl substance (PFAS) and deflects, the laser light is reflected at a slightly different angle, changing the way the reflected laser spot reaches each quadrant and thus the value of AK AEis proportional to microcantilever deflection. By measuring AF, the system monitors the

[0065] 10

[0066] QB\702581.02730\100020683.1 deflection of the cantilever in real-time (<0.1 ms readout time) and with high precision (< 01 nm accuracy).

[0067] In some embodiments, the system is configured as a packaged and fieldable detection platform including a cartridge containing at least one microcantilever (i.e., a microcantilever cartridge), and a portable reader. In some cases, the microcantilever cartridge includes one or more polypeptide functionalized microcantilevers. Optionally, the microcantilever cartridge may include one or more reference microcantilevers for differential readout to eliminate any false positives. The microcantilever cartridge may be configured to interface with a sample handling module. In some cases, the sample handling module includes a system configured to introduce a liquid sample to the microcantilevers, optionally including filtration, mixing, dilution, and / or buffer exchange. In some cases, the microcantilever cartridge is configured for single-use or limited reuse and comprises fluidic interfaces suitable for coupling to a handheld or benchtop reader. In some cases, the sample handling module may include microfluidics operations.

[0068] In some embodiments, the portable reader includes: (i) an optical detection module configured to detect microcantilever deflection and / or an electrical detection module configured to detect microcantilever deflection using integrated electronics; (ii) a computing platform configured to receive signals from the detector and determine the response of the microcantilever(s) to the sample; and (iii) a user interface. In some cases, the computing platform is configured to provide a result indicating the presence or absence of a PFAS and / or an estimated concentration of the PFAS. In some cases, the system is configured for multiplexed detection using two or more cantilevers functionalized with different polypeptides.

[0069] In some embodiments, the system further comprises a computing platform, shown in FIG. 7, having a communication interface that receives one or more signals from the detector, and a computer in communication with the communication interface, wherein the computer comprises a computer processor and a computer readable medium comprising machine-executable code that, upon execution by the computer processor, implements a method for determining the response of the one or more nanomechanical sensors to the sample.

[0070] The computational platform is capable of characterizing and determining the response of one or more nanomechanical sensors to the sample. The computational platform may generally include various input / output (I / O) modules, one or more processing units, a memory, and a communication network.

[0071] 11

[0072] QB\702581.02730\100020683.1 In some implementations, the computational platform may be any general -purpose computing system or device, such as a personal computer, workstation, cellular phone, smartphone, laptop, tablet, or the like. In this regard, the computational platform may be a system designed to integrate a variety of software, hardware, capabilities, and functionalities. Alternatively, and by way of configurations and programming, the computational platform may be a special-purpose system or device.

[0073] The computational platform may operate autonomously or semi-autonomously based on user input, feedback, or instructions. In some implementations, the computational platform may operate as part of, or in collaboration with, various computers, systems, devices, machines, mainframes, networks, and servers. For instance, the computational platform may communicate with one or more servers or databases, by way of a wired or wireless connection.

[0074] The VO modules of the computational platform may include various input elements, such as a mouse, keyboard, touchpad, touchscreen, buttons, microphone, and the like, for receiving various selections and operational instructions from a user. The I / O modules may also include various drives and receptacles, such as flash-drives, USB drives, CD / DVD drives, and other computer-readable medium receptacles, for receiving various data and information. To this end, I / O modules may also include a number of communication ports and modules capable of providing communication via Ethernet, Bluetooth, or Wi-Fi, to exchange data and information with various external computers, systems, devices, machines, mainframes, servers, networks, and the like. In addition, the VO modules may also include various output elements, such as displays, screens, speakers, LCDs, and others.

[0075] The processing unit(s) may include any suitable hardware and components designed or capable of carrying out a variety of processing tasks, including steps implementing the present framework for response detection. To do so, the processing unit(s) may access or receive one or more signals generated by the detector. The signals may be stored or tabulated in the memory, in the storage server(s), in the database(s), or elsewhere. In addition, such information may be provided by a user via the I / O modules or selected based on user input.

[0076] In some configurations, the processing unit(s) may include a programmable processor or combination of programmable processors, such as central processing units (CPUs), graphics processing units (GPUs), and the like. In some implementations, the processing unit(s) may be configured to execute instructions stored in a non-transitoiy computer readable-media of the

[0077] 12

[0078] QB\702581.02730\100020683.1 memory. The non-transitory computer-readable media may be included in the memory, it may be appreciated that instructions executable by the processing unit(s) may be additionally, or alternatively, stored in another data storage location having non-transitory computer-readable media.

[0079] In some configurations, the processing unit(s) may include one or more dedicated processing units or modules configured (e.g., hardwired, or pre-programmed) to carry out steps, in accordance with aspects of the present disclosure. Each solver module may be configured to perform a specific set of processing steps, or carry out a specific computation, and provide specific results.

[0080] The processing unit(s) may also be configured to generate a report and provide it via the I / O modules. The report may be in any form and provide various information. For instance, the report may include various numerical values, text, graphs, maps, images, illustrations, and other renderings of information and data. In particular, the report may provide various information or properties generated by the processing unit(s) from the detector generated signals. The report may also include various metrics or indices for determining whether a PFAS molecule is present in a sample. To this end, the report may be provided to a user, or directed via the communication network to an assembly line or various hardware, computers, or machines therein.

[0081] Referring to Figure 3, disclosed herein is a system for detecting a per- and polyfluoroalkyl substance (PFAS), including: a first microcantilever (100); and one or more polypeptides (200) coupled to at least a portion of the first microcantilever, wherein interaction of the one or more polypeptides with the per- and polyfluoroalkyl substance (PFAS) induces a detectable deflection in the first microcantilever. In some cases, the system may further include a second microcantilever (101) one or more of a second polypeptide (201) coupled to at least a portion of the second microcantilever, wherein interaction of each of the one or more second polypeptides with a second per- and polyfluoroalkyl substance (PFAS) induces a deflection in the second microcantilever. The one or more second polypeptides (201) are distinct from the one or more first polypeptides (200) coupled to at least a portion of the first microcantilever (100). In some cases, the system may include a control microcantilever (102) substantially-free of one or more polypeptides. The system may include a detector system (300), including optical detection and / or electrical detection as described above. The detection system may communicate with a processor (302).

[0082] 13

[0083] QB\702581.02730\100020683.1 Referring to Figure 4, disclosed herein is a method for detecting a per- and polyfluoroalkyl substance (PFAS), including: exposing (400) a sample containing a per- and polyfluoroalkyl substance (PFAS) to a first polypeptide functionalized microcantilever (3) including a first microcantilever, and one or more polypeptides coupled to at least a portion of the first microcantilever, wherein interaction of the one or more polypeptides with the per- and polyfluoroalkyl substance (PFAS) induces a detectable deflection in the first microcantilever; and detecting (500) a movement of the microcantilever. In some cases, the methods may include exposing a sample containing per- and polyfluoroalkyl substances (PFAS) to a second polypeptide functionalized microcantilever including a second microcantilever, and one or more of a second polypeptide coupled to at least a portion of the second microcantilever, wherein each of the one or more second polypeptides interacts with a per- and polyfluoroalkyl substance (PFAS), and wherein the one or more second polypeptides is distinct from the respective one or more polypeptides coupled to at least a portion of the microcantilever; and detecting a movement of the second microcantilever. The method may further include comparing (600) the movement of the first polypeptide functionalized microcantilever and / or the movement of the second polypeptide functionalized microcantilever to the movement of a control microcantilever substantially-free of one or more polypeptides.

[0084] The methods described herein allow for rapid detection of the target. In some embodiments, the response to the target antigen or the target antibody is detected within 1 second, 10 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, or longer of contacting the microcantilever with the sample.

[0085] Disclosed herein are detectable complexes including a polypeptide including a binding site and exposed amino acid residues; and a per- and polyfluoroalkyl substance (PFAS). The PFAS includes a fluoroalkyl tail and a polar terminal group. The detectable complex includes the fluoroalkyl tail and a polar terminal group within the binding site and a polar terminal group interacting with the exposed amino acid residues. The polypeptide may be immobilized on a substrate, such as a microcatelever.

[0086] In another aspect, the detectable complexes disclosed herein include a per- and polyfluoroalkyl substance (PFAS). the PFAS is selected from the group consisting of perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), perfluoroocatanesulfonate (PFOS), perfluorooctanesulfonamide (PFOSA), perfluorononanoic acid (PFNA),

[0087] 14

[0088] QB\702581.02730\100020683.1 perfluorodecanoic acid (PFDA), perfluorohexane sulfonic acid (PFHxS), and any combinations thereof.

[0089] In some cases, the binding site may include a hydrophobic binding site or an amphipathic binding site. In another aspect, the exposed amino acid residue may include aromatic residues. In some cases, the exposed amino acid residue may be nonaromatic residues. The polar terminal group interacting with the exposed amino acid residues may include directional hydrogen bonding. In some cases, the polar terminal group interacting with the exposed amino acid residues may include electrostatic interactions.

[0090] The polypeptide may be immobilized on a substrate. The substrate may be a microcantilever but it need not be. The detectable complexes disclosed herein may further include one or more components of a self-assembled monolayer (SAM) on the substrate. In some cases, the SAM may include 11-mercaptoundecanoic acid (MUA). Optionally, the detectable complexes may further include a spacer. In some cases, the spacer may be positioned between the SAM and the polypeptide. In some cases, the spacer may include an amine. In one example, 3-aminophenylboronic acid (APB A) may be the spacer.

[0091] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0092] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0093] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0094] 15

[0095] QB\702581.02730\100020683.1 All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0096] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0097] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0098] EXAMPLES

[0099] Example 1

[0100] Detecting PFOS (Perfluorooctane Sulfonic Acid) and PFOA (Perfluorooctanoic Acid) in environmental and biological samples is crucial due to their widespread presence and potential health risks, but challenges exist in detecting PFOS and PFOA at very low concentrations.

[0101] Microcantilever Detection of PFOA and PFOS using Nano-electro-mechanical Technology

[0102] Microcantilever sensors provide a highly sensitive and innovative approach for detecting PFAS, such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), in diverse environments. These sensors operate by detecting mechanical changes caused by the adsorption of target molecules onto the cantilever surface. A microcantilever sensor typically consists of a small, thin cantilever beam anchored at one end, with the other end free to move. The

[0103] 16

[0104] QB\702581.02730\100020683.1 surface of the microcantilever is functionalized with a coating or layer that selectively binds to the target molecules. In static mode, the adsorption of target molecules causes a surface stress change, leading to the bending or deflection of the cantilever. Microcantilever sensors offer several advantages. They exhibit high sensitivity, are capable of detecting minute changes in mass or surface stress, and allow for the detection of low concentrations of PFAS. These sensors provide real-time detection, making them suitable for continuous monitoring applications. Additionally, microcantilever sensors do not require labeling or complex sample preparation, simplifying the detection process.

[0105] Selection of Antibodies for detection of PFOA and PFOS on microcantilever

[0106] The detection of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) can be significantly enhanced by using specific biomarkers and antibodies. Liver-type fatty acidbinding protein (L-FABP) and antialbumin antibodies have proven to be particularly effective in this context. L-FABP is a biomarker that binds to fatty acids and other hydrophobic molecules, including PFOA and PFOS. Its high affinity for these substances makes it a valuable tool for detecting them in biological samples. Similarly, anti-albumin antibodies target albumin, a common protein in blood that can bind to various substances, including PFOA and PFOS. These antibodies can isolate and detect albumin-bound PFOA and PFOS in biological samples. Therefore, we selected the above mentioned two antibodies to test the detection of several PFAS derivatives, including Heptafl orobutyric acid (HFBA), Perflurooctonoic acid (PFOA), and Heptadecafluorooctanesulfonic acid potassium salt (PFSA).

[0107] Functionalization of microcantilevers

[0108] The cantilevers were immersed in deionized water followed by isopropanol, both purchased from Merck Millipore, for about 60 minutes at each step. They were then washed with deionized water. Before functionalization, the gold-coated cantilevers were plasma cleaned to remove any potential contaminants prior to the antibody immobilization step, antibodies (L-FABP or anti-albumin) were covalently attached to the gold-coated cantilever surface through EDC-NHS coupling chemistry inside a glass plate containing tiny wells. A 200 pL DPBS solution was added to the wells containing the microcantilevers for functionalization and incubated for about 5 minutes. After 5 minutes, the DPBS solution was carefully removed using a micropipette. To form

[0109] 17

[0110] QB\702581.02730\100020683.1 the self-assembled monolayer (SAM) on the cantilever surface via thiol-metal (gold) bonding, the cantilevers were immersed in 200 pL of a 10 mM solution of 11-Mercaptoundecanoic acid (MU A) prepared in ethanol for about 30 minutes, followed by rinsing three times with deionized water. The carboxyl anions on the SAM surface were then activated by adding a 1 : 1 ratio of a 100 pL solution mixture of 5 mM Carbodiimide EDC and 5 mM Sulfo-NHS in deionized water to attach the amino radicals of other biomolecules via amide linkage. The cantilevers were left for incubation for about 40 minutes. After 40 minutes, the cantilevers were rinsed three times with deionized water and submerged in a 50 pL solution of 52 mM APBA (3-aminophenylboronic acid monohydrate, 98%) (10 mM PBS at pH 7.4) for 3 hours. After 3 hours, the SAM-functionalized microcantilevers were rinsed three times with DPBS buffer and incubated overnight at 4°C in 100 pL of 20 pg / mL antibody (L-FABP) or 2 pg / mL (anti-albumin antibody) solutions prepared in DPBS and 0.05% bovine serum albumin (pH 7.4) to facilitate covalent immobilization. Bovine serum albumin (BSA) was used to block the remaining sensor surface to minimize non-specific interactions. The optimization of antibody concentration was concluded based on good deflection measurements taken during a series of experiments against various concentrations of PF As and antibody. Before performing real-time assay experiments, the microcantilevers were washed with PBS-Tween-20 solution, dried, and then fixed in the AFM sample holder.

[0111] Detection studies of HFBA, PFOA and PFSA on L-FABP antibody coated microcantilever respectively.

[0112] A set of different concentrations of HFBA, PFOA, and PFSA were used for deflection studies on L-FABP coated microcantilevers. Initial screening of the deflection studies for HFBA and cantilever immobilization was performed according to the following experimental protocols. Microcantilevers immobilized with L-FABP antibody (20 pg / ml) in triplicate using EDC-NHS chemistry were used to quantify the detection signal for various HFBA concentrations (1.0 ppm, 0.01 ppm, 100 ppt, 10 ppt, and 1.0 ppt) in buffered solution to study the deflection kinetics. The cantilever with the highest concentration (1.0 ppm) showed the maximum deflection (53.11 nm), whereas the lowest concentration (1.0 ppt) showed the least deflection (16.51 nm) as shown in Figure la. For PFOA and PFSA, concentrations of 10 ppm and 100 ppm were used to study the deflection on L-FABP coated microcantilevers. The cantilever with the highest concentration (100 ppm) of PFOA and PFSA showed deflections of 73.91 nm and 1.51 nm, respectively, while the

[0113] 18

[0114] QB\702581.02730\100020683.1 cantilever with the lowest concentration (10 ppm) showed deflections of 58.46 nm for PFOA and 1.23 nm for PFSA, as depicted in Figure lb.

[0115] Table 1: Maximum deflection data of PFAs on L-FABP antibody coated microcantilevers.

[0116] Detection studies of HFBA, PFOA and PFSA on anti-albumin antibody coated microcantilever respectively.

[0117] A set of different concentrations of HFBA, PFOA, and PFSA were used for deflection studies on anti-albumin coated microcantilevers. Initial screening of the deflection studies for HFBA and cantilever immobilization was performed according to the following experimental protocols. Microcantilevers immobilized with anti-albumin antibody (2 pg / mL) in triplicate using EDC-NHS chemistry were used to quantify the detection signal for various HFBA concentrations (100 ppt, 10 ppt, and 1.0 ppt) to study the deflection kinetics. The cantilever with the highest concentration (100 ppt) showed the maximum deflection (27.21 nm), whereas the lowest concentration (1.0 ppt) showed the least deflection (11.09 nm), as shown in Figure 2a. For PFOA and PFSA compounds, concentrations of 10 ppm and 100 ppm were used for deflection studies on 19

[0118] QB\702581.02730\100020683.1 anti-albumin coated microcantilevers. The cantilever with the highest concentration (100 ppm) of PFOA and PFSA showed deflections of 51.91 nm and 2.11 nm, respectively, while the cantilever with the lowest concentration (10 ppm) showed deflections of 32.56 nm for PFOA and 1.99 nm for PFSA, as depicted in Figure lb.

[0119] Table 2: Maximum deflection data of PFAs on anti-albumin antibody coated microcantilevers.

[0120] Conclusion Coated microcantilever sensors, such as the exemplary L-FABP or anti-albumin coated microcantilever sensors disclosed herein, represent a promising technology for the detection of PFOA and PFOS, offering high sensitivity, real-time monitoring, and label-free detection. Ongoing research and development aim to address challenges related to selectivity, sensitivity, and reproducibility, making these sensors increasingly viable for environmental monitoring, industrial waste assessment, and public health applications.

[0121] 20

[0122] QB\702581.02730\100020683.1 Example 2

[0123] Disclosed herein are detectable complexes including a polypeptide including a binding site and exposed amino acid residues; and a per- and polyfluoroalkyl substance (PFAS). Aspects of the detectable complexes are described below.

[0124] Proteins involved in fatty-acid transport — including human serum albumin (HSA) and liver-type fatty-acid binding protein (L-FABP) — exhibit high-affinity interactions with PFAS through hydrophobic and van der Waals forces between perfluorinated tails and nonpolar domains. Harnessing these interactions on engineered microcantilever surfaces enables selective, rapid, label-free nanomechanical transduction via surface-stress-induced deflection.

[0125] Multi-Point Ligand-Receptor Complex Formation

[0126] PFAS molecules bind through a dual-domain interaction mechanism, which includes the perfluoroalkyl tail inserting into a hydrophobic or amphipathic pocket within the antibody (e.g., mimicking L-FABP / albumin PFAS binding motifs), and the polar headgroup (carboxylate or sulfonate) forming directional hydrogen-bonding or electrostatic interactions with exposed amino acid residues. This generates a multi-point anchoring geometry, leading to a tightly bound PFAS- biological ligand-linker (e.g., PFAS-antibody-linker, PFAS-Ab-SAM) complex with restricted rotational freedom.

[0127] Molecular Packing and Interlayer Reorganization

[0128] PFAS binding induces local crowding among adjacent immobilized antibodies, forcing rearrangement of antibody Fab domains, compaction or spreading of the SAM / linker layer [MUA (11-Mercaptoundecanoic Acid) or MUA-APBA], and displacement of tightly bound interfacial water and counter-ions. These rearrangements increase lateral intermolecular forces and generate a coherent compressive stress field across the functionalized surface.

[0129] Induced Conformational Changes in the Antibody Layer

[0130] Antibody-PFAS binding is accompanied by conformational movements such as loop tightening around the PFAS chain, hinge-region compaction, and minor reorientation of variable domains upon ligand capture. When many antibodies undergo these changes in parallel on a

[0131] 21

[0132] QB\702581.02730\100020683.1 densely grafted monolayer, the collective conformational transitions generate a measurable net surface free-energy change, manifested as compressive stress.

[0133] Stoichiometry-Driven Amplification of Mechanical Stress

[0134] Each PFAS molecule forms a compact, low-entropy complex, which produces contraction of the antibody-SAM interface, increased packing density, and creation of a lateral compressive force vector parallel to the cantilever plane. Accumulation of many such complexes across hundreds of nanometers produces sufficient net compressive stress to bend the cantilever, enabling label-free nanomechanical transduction.

[0135] Mechanical Transduction Advantage Over Electrostatic Sensors

[0136] Because signaling originates from surface stress generated by conformational and packing changes, rather than charge transfer, the PFAS-antibody complex is insensitive to high ionic strength, avoids electrostatic screening failure, and maintains high fidelity in environmental water samples.

[0137] Disclosed are devices, systems, and methods employing microcantilever sensors whose gold surfaces are functionalized with self-assembled monolayers (SAMs) and covalently immobilized antibodies that recognize PFAS through biomolecular mimicry of fatty -acid binding. In exemplary embodiments, an 11-mercaptoundecanoic acid (MU A) SAM is activated by EDC / NHS, optionally coupled to 3-aminophenylboronic acid (APB A) as an amine-bearing spacer, and used to immobilize anti-L-FABP and / or anti-albumin antibodies. Upon exposure to a sample, PFAS binding at the antibody layer generates compressive differential surface stress, deflecting the cantilever in real time.

[0138] Selective capture is achieved through (i) protein-ligand complementarity that favors PFAS over non-PFAS interferents; (ii) speciation control (e.g., free-acid vs salt forms) that preserves epitope presentation; (iii) antifouling blocking (BSA) and buffer choices; and (iv) ratiometric referencing using non-functionalized or isotype-control cantilevers. The platform detects multiple PFAS within minutes at ultra-low concentrations and is inherently robust to ionic screening compared with charge-based FET sensors because transduction is mechanical rather than electrostatic.

[0139] 22

[0140] QB\702581.02730\100020683.1 Example 3

[0141] Mechanism of Specific Capture

[0142] Selectivity arises from biomolecular recognition between PFAS and antibody surfaces that present epitopes mimicking the natural PFAS-protein interactome. Anti-L-FABP exposes hydrophobic pockets and aromatic residues favorable for perfluoroalkyl chains, enabling strong, broad-spectrum PFAS capture. Anti-albumin exhibits selective binding patterns consistent with albumin's role as a PFAS carrier, and responses differ across PFAS chemotypes. Speciation control (free acid vs salt) modulates epitope presentation, where PFOS (free acid) is captured, whereas PFOS potassium salt shows minimal response under identical conditions, evidencing chemical-state specificity. Antifouling and buffer control suppress non-specific adsorption and electrostatic artifacts.

[0143] Because the transduction is mechanical, signal fidelity is less sensitive to ionic strength and background charges than electrostatic FET sensors, improving robustness in complex water matrices.

[0144] Example 4

[0145] FIG. 5 and FIG. 6 provide dose-dependent detection of PFA derivatives using anti-albumin antibody immobilized microcantilever platform and anti-L-FABP antibody immobilized microcantilever platform, respectively. Summarized below are maximum deflection values in nanometers for the highest concentration of PFA derivative tested (lO ng / mL) and the lowest concentration of PFA derivative tested (lOO fg / mL). Deflection values for buffer controls are provided. Buffer-only conditions are used as control conditions, where the buffer is processed in the same manner as PF AS-containing samples and contacted with the functionalized microcantilever for the same duration and under the same measurement conditions. Reference microcantilevers are used as control sensors for differential measurements, including cantilevers that are non-functionalized or functionalized substantially the same as working polypeptide functionalized microcantilevers and are further treated to prevent PFAS molecule interactions. For example, the reference microcantilevers may be functionalized with polypeptides treated (i.e., ‘treated polypeptides’) which are incapable of PFAS binding observed in working polypeptide functionalized microcantilevers. In some cases, BSA blocking and buffer selection reduce nonspecific interactions, and differential subtraction of reference cantilever response improves

[0146] 23

[0147] QB\702581.02730\100020683.1 selectivity and robustness. Inventors observed that, because transduction is mechanical (surface stress-induced deflection) rather than electrostatic, the detection signal exhibits improved robustness to ionic strength and electrostatic screening in complex sample matrices. In some embodiments, selectivity is supported by binder-dependent response differences across PFAS chemotypes and / or PFAS speciation, and by use of reference cantilevers and / or buffer controls as described herein.

[0148] L-FABP -Functionalized Cantilevers

[0149] For HFBA, the maximum deflection ranged from approximately -39.12 to -9.96 nm across concentrations from 10 ng / mL to 100 fg / mL, with buffer showing approximately -1.51 nm. PFOA exhibited deflections of -37.39 to -10.34 nm. PFOS (free acid) showed deflections of -28.53 to -6.27 nm, while PFOSA demonstrated deflections of -40.23 to -5.07 nm. The limit of detection (LOD) was at least 100 fg / mL, detectable within <3 min across multiple PFAS in a dose-dependent manner.

[0150] Anti-Albumin-Functionalized Cantilevers

[0151] For HFBA, deflections ranged from -35.48 to -7.84 nm, with buffer showing approximately +1.06 nm. PFOA exhibited deflections of -34.51 to -6.87 nm, with buffer showing approximately -2.72 nm. PFOS (free acid) showed deflections of -16.57 to -2.77 nm, which was attenuated relative to L-FABP. PFOSA demonstrated deflections of -28.69 to -7.97 nm.

[0152] Regarding selectivity, PFOS potassium salt elicited approximately 1-2 nm deflection at 10-100 ppm on L-FABP and approximately 2 nm on anti-albumin. In comparison, the free-acid PFOS and other PFAS showed deflections on the order of tens of nm, indicating chemi cal -state selectivity.

[0153] Example 5

[0154] Comparative Analysis of PFAS Interactions with Anti-Albumin and Anti-L-FABP Antibodies

[0155] Anti-L-FABP antibody consistently produced larger, more concentration-dependent deflection signals for all PFAS analytes compared to anti-albumin, indicating stronger binding 24

[0156] QB\702581.02730\100020683.1 affinity and higher analytical sensitivity. L-FABP maintained strong and distinguishable responses across all four PFAS compounds tested (HFBA, PFOA, PFOS, PFOSA), whereas anti-albumin showed selective preference only for HFBA and PFOA at higher concentrations and performed modestly with PFOS. L-FABP retained measurable and meaningful signal output even at lOO fg / mL, particularly for PFOA, demonstrating ultra-low detection limits suitable for environmental and biological monitoring.

[0157] Anti -L-FABP showed a clear concentration-dependent trend from lO ng / mL down to lOO fg / mL, whereas anti-albumin had less sensitivity rapidly at lower concentrations, with PFOS showing the weakest binding. L-FABP provided high responses for structurally diverse PF AS molecules (short-chain, long-chain, sulfonates, and acids), suggesting stronger versatility as a recognition element. The large signal windows produced by L- FABP, e g., substantial deflections for PFOSA and PFOA at 10 ng / mL, support improved quantitative accuracy and lower false-negative likelihood.

[0158] The performance of L-FABP indicates that it is a suitable antibody for next- generation NEMS / microcanti lever-based PF AS sensors, especially for applications demanding ultra-trace detection. L-FABP's ability to detect PFAS at fg / mL levels aligns directly with contamination levels observed in real-world water samples and biological matrices, enhancing translational significance.

[0159] 25

[0160] QB\702581.02730\100020683.1

Claims

CLAIMSWhat is claimed is:

1. A system for detecting a per- and polyfluoroalkyl substance (PFAS), comprising: a first microcantilever; and one or more polypeptides coupled to at least a portion of the first microcantilever, wherein interaction of the one or more polypeptides with the per- and polyfluoroalkyl substance (PFAS) induces a detectable deflection in the first microcantilever.

2. The system of claim 1, wherein contact of a sample comprising less than 100 ppm, less than 10 ppm, less than 1 ppm, less than 100 ppt, less than 10 ppt, or about 1 ppt of the PFAS with the one or more polypeptides induces a detectable deflection in the first microcantilever.

3. The system of claim 1, wherein the per- and polyfluoroalkyl substances (PFAS) is a perfluoroalkyl carboxylic acid.

4. The system of claim 3, wherein the PFAS is selected from the group consisting of perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), perfluoroocatanesulfonate (PFOS), perfluorooctanesulfonamide (PFOSA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorohexane sulfonic acid (PFHxS), and any combinations thereof.

5. The system of any one of claims 1-4, wherein the one or more polypeptides are one or more antibodies.

6. The system of claim 5, wherein the one or more antibodies comprises liver-type fatty acidbinding protein, antialbumin antibodies, and any combinations thereof.

7. The system of claim 5, wherein the one or more antibodies comprises liver-type fatty acidbinding antibody (anti-L-FABP).26QB\702581.02730\100020683.

18. The system of claim 5, wherein the one or more antibodies comprises anti-albumin antibody.

9. The system of any one of claims 1-8, wherein the first microcantilever is at least partly coated in gold.

10. The system of any one of claims 1-9, wherein one or more polypeptides coupled to at least a portion of the first microcantilever are covalently attached to the first microcantilever.

11. The system of claim 10, wherein the one or more polypeptides coupled to at least a portion of the first microcantilever are covalently attached to the first microcantilever by thiol-metal and / or l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) coupling.

12. The system of any one of claims 1-11, wherein the first microcantilever further comprises a surface area substantially-free of polypeptides; and wherein the surface area substantially-free of polypeptides is coupled to bovine serum albumin.

13. The system of any one of claims 1-12, wherein the first microcantilever has a maximum dimension of 100 pm or more, 1.5 mm or less, or of from about 100 pm to about 600 pm.

14. The system of any one of claims 1-13, further comprising: a second microcantilever; one or more of a second polypeptide coupled to at least a portion of the second microcantilever, wherein interaction of each of the one or more second polypeptides with a second per- and polyfluoroalkyl substance (PFAS) induces a deflection in the second microcantilever; and wherein the one or more second polypeptides are distinct from the one or more polypeptides coupled to at least a portion of the first microcantilever.

15. The system of any one of claims 1-14, further comprising a control microcantilever.27QB\702581.02730\100020683.

116. The system of any one of claims 1-15, wherein the first microcantilever has an effective surface density of coupled polypeptides thereon.

17. The system of claim 14, wherein the second microcantilever is a control microcantilever.

18. The system of claim 17, wherein the control microcantilever comprises one or more blocked polypeptides coupled to at least a portion of the reference microcantilever.

19. A method for detecting a per- and polyfluoroalkyl substance (PF AS), comprising: exposing a sample containing a per- and polyfluoroalkyl substance (PFAS) to a first polypeptide functionalized microcantilever comprising: a first microcantilever, and one or more polypeptides coupled to at least a portion of the first microcantilever, wherein interaction of the one or more polypeptides with the per- and polyfluoroalkyl substance (PFAS) induces a detectable deflection in the first microcantilever; and detecting a movement of the first microcantilever.

20. The method of claim 19, wherein the one or more polypeptides are one or more antibodies.

21. The method of claim 20, wherein the one or more antibodies comprises liver-type fatty acid-binding protein, antialbumin antibodies, and any combinations thereof.

22. The method of claim 20, wherein the one or more antibodies comprises liver-type fatty acid-binding antibody (anti-L-FABP).

23. The method of claim 20, wherein the one or more antibodies comprises anti-albumin antibody.

24. The method of any one of claims 19-23, wherein detecting the movement of the first microcantilever comprises utilizing an optical detection method and / or an electrical detection method.28QB\702581.02730\100020683.

125. The method of any one of claims 19-24, wherein detecting the movement of the first microcantilever comprises optical detection using reflected light and a position sensitive detector.

26. The method of any one of claims 19-24, wherein detecting a movement of the first microcantilever comprises electrical detection using an integrated field effect transistor (FET), metal oxide semiconductor (MOSFET), or bipolar metal oxide (BiMOS) transistor.

27. The method of any one of claims 19-26, wherein the per- and polyfluoroalkyl substances (PFAS) bind to the one or more polypeptides.

28. The method of any one of claims 19-27, wherein exposing the per- and polyfluoroalkyl substances (PFAS) in the sample to the first polypeptide functionalized microcantilever results in detectable movement of the first microcantilever within 10 seconds, within 30 seconds, within 1 minute, within 2 minutes, within 3 minutes, within 5 minutes, or within 10 minutes from the exposing the sample to the first microcantilever.

29. The method of any one of claims 19-28, wherein the one or more polypeptides comprise liver-type fatty acid-binding protein or antialbumin antibodies, and wherein the per- and polyfluoroalkyl substance (PFAS) comprises a perfluoroalkyl carboxylic acid.

30. The method of claim 29, wherein the perfluoroalkyl carboxylic acid is selected from the group consisting of perfluorooctanoic acid, heptafluorobutyric acid, and any combinations thereof.

31. The method of any one of claims 19-30, wherein the sample comprises less than 100 ppm, less than 10 ppm, less than 1 ppm, less than 100 ppt, less than 10 ppt, or about 1 ppt of the per- and polyfluoroalkyl substance (PFAS).

32. The method of any one of claims 19-31, wherein the first microcantilever is present in a microfluidic system.29QB\702581.02730\100020683.

133. The method of any one of claims 19-32, further comprising: exposing the sample to a second polypeptide functionalized microcantilever comprising: a second microcantilever, and one or more of a second polypeptide coupled to at least a portion of the second microcantilever, wherein each of the one or more second polypeptides interacts with a per- and polyfluoroalkyl substance (PFAS), and wherein the one or more second polypeptides is distinct from the respective one or more polypeptides coupled to at least a portion of the microcantilever; and detecting a movement of the second microcantilever.

34. The method of anyone of claims 19-33, further comprising comparing the movement of the first polypeptide functionalized microcantilever and / or the movement of the second polypeptide functionalized microcantilever to the movement of a control microcantilever.

35. A detectable complex, comprising: a polypeptide comprising a binding site and an exposed amino acid residue; and a per- and polyfluoroalkyl substance (PFAS) comprising: a fluoroalkyl tail and a polar terminal group within the binding site; and a polar terminal group interacting with the exposed amino acid residues.

36. The detectable complex of claim 35, wherein the peptide is immobilized on a surface.

37. The detectable complex of any one of claims 35-36, wherein the binding site comprises a hydrophobic binding site or an amphipathic binding site.

38. The detectable complexes of any one of claims 35-37, wherein the exposed amino acid residue comprise aromatic residues.

39. The detectable complex of any one of claims 35-38, wherein the polar terminal group interacting with the exposed amino acid residues comprises directional hydrogen bonding or electrostatic interactions.30QB\702581.02730\100020683.

140. The detectable complex of any one of claims 35-39, further comprising a self-assembled monolayer (SAM).

41. The detectable complex of claim 40, wherein the SAM comprises 11-mercaptoundecanoic acid (MU A).

42. The detectable complexes of any one of claims 35-41, wherein the polypeptide comprises an antibody.

43. The detectable complexes of claim 42, wherein the antibody comprises liver-type fatty acid-binding protein (anti-L-FABP), anti-albumin antibodies, and any combinations thereof.

44. The detectable complexes of any one of claims 35-43, wherein the fluoroalkyl tail is selected from the group consisting of perfluorobutyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluor ononyl, perfluorodecyl, and any combinations thereof.

45. The detectable complexes of any one of claims 35-44, wherein the terminal polar group is selected from the group consisting of carboxylic acid, carboxylate, sulfonic acid, sulfonate, phosphonic acid, phosphonate, phosphinic acid, phosphinate, alcohol, sulfonamide, ether, amide, and any combinations thereof.

46. The detectable complexes of any one of claims 35-45, wherein the PFAS is selected from the group consisting of perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), perfluoroocatanesulfonate (PFOS), perfluorooctanesulfonamide (PFOSA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorohexane sulfonic acid (PFHxS), and any combinations thereof.31QB\702581.02730\100020683.1

Images