Systems and methods for electrically detecting a biomarker

Abstract

A system for electrically detecting a biomarker, the system comprising: a sample unit comprising: a sample chamber configured to contain a fluid sample; a reference electrode in fluid communication with the sample chamber; a detection zone in fluid communication with the sample chamber and configured to capture a biomarker; a signal zone in fluid communication with the detection zone; and a working electrode configured such that its surface potential changes in the presence of charged species (e.g., protons) generated by a reaction (e.g., between an enzyme and a radical substrate) occurring in the signal zone; and a measuring unit comprising a field-effect transistor in electrical communication with the reference electrode and the working electrode, the measuring unit configured to measure a change in surface potential of the working electrode due to generated charged species.

Description

SYSTEMS AND METHODS FOR ELECTRICALLY DETECTING A BIOMARKERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. provisional patent application number 63 / 638,023 that was filed April 24, 2024. the entire contents of which are incorporated herein by reference.BACKGROUND

[0002] Healthcare systems worldwide are grappling with the demands of aging populations and increasingly complex health needs, exacerbated by workforce shortages, capacity constraints, and rising costs. In this context, home-based care has gained prominence, offering high patient satisfaction, cost-effective management, and reduced readmission rates, all while maintaining healthcare quality. Shifting toward home-based healthcare requires accurate, low-cost, rapid, and portable point-of-care (POC) clinical testing to manage a variety of conditions. A relevant application is monitoring fertility7hormones such as estradiol (E2). High E2 levels can contribute to conditions such as fibroids, endometriosis, and increased cancer risk. Conversely, low E2 levels may indicate ovarian insufficiency or menopause, impacting fertility and reproductive health. Current hormone assays for estrogen involve lab-based chemiluminescence immunoassays due to its low and variable concentration during pregnancy (20 pg / mL to 20,000 pg / mL) and susceptibility to biomolecular interference. Crucially, extremely low concentrations of E2 under a few pg / mL, relevant for non-reproductive issues, challenge current analytical methods due to difficulties in achieving accurate measurements.SUMMARY

[0003] Provided are systems and methods for electrically detecting a biomarker (e.g., estradiol) in a test specimen (e.g., blood, serum, plasma, saliva, urine, sweat, etc.) of a subject (e.g., a mammalian subject). The systems and methods are not only sensitive and specific but also widely accessible, enabling monitoring of crucial biomarkers without the prohibitive costs or logistical challenges of current methodologies.

[0004] The present systems and methods are illustrated by reference to an Example, below, that demonstrates a radical-mediated enzyme assay (REEA) that integrates with a paper fluidic system and is electrically read by a handheld field-effect transistor (FET)device. The REEA utilizes horseradish peroxidase (HRP) to catalyze the conversion of aromatic substrates into radical forms, producing protons detected by a FET for biomarker quantification. Through screening 14 phenolic compounds, halogenated phenols emerged as optimal substrates for the REEA. Encased in an affordable cartridge ($0.55 per test), the system achieved a detection limit of 146 fg / mL for estradiol (E2), with a coefficient of variation (CV) below 9.2% in E2-spiked buffer samples and an accuracy (r2) of 0.963 across a measuring range of 19 to 4,551 pg / mL in clinical plasma samples, providing results in under 10 minutes. This adaptable system not only provides a fast and reliable platform, but may also be used with a wide array of biomarkers, enabling clinical and home-based applications for a broad range of health conditions.

[0005] An embodiment 1 is a system for electrically detecting a biomarker, the system comprising: a sample unit comprising: a sample chamber configured to contain a fluid sample; a reference electrode in fluid communication with the sample chamber; a detection zone in fluid communication with the sample chamber and configured to capture a biomarker; a signal zone in fluid communication with the detection zone; and a working electrode configured such that its surface potential changes in the presence of charged species generated by a reaction occurring in the signal zone; and a measuring unit comprising a fieldeffect transistor in electrical communication with the reference electrode and the working electrode, the measuring unit configured to measure a change in surface potential of the working electrode due to generated charged species.

[0006] An embodiment 2 is according to embodiment 1, wherein the charged species comprise protons and the reaction is between an enzyme and a radical substrate.

[0007] An embodiment 3 is according to any of embodiments 1-2, wherein the detection zone comprises immobilized secondary antibodies for capturing a primary antibody of the biomarker.

[0008] An embodiment 4 is according to embodiment 3, wherein the biomarker comprises a hormone, a protein, a chemical, a biological agent, a virus, or a bacteria.

[0009] An embodiment 5 is according to embodiment 3, wherein the biomarker comprises a fertility hormone.

[0010] An embodiment 6 is according to any of embodiments 2-5, wherein the signal zone is configured to induce the reaction between the enzyme and the radical substrate.

[0011] An embodiment 7 is according to embodiment 6, wherein the signal zone comprises immobilized enzymes that produce a peroxide.

[0012] An embodiment 8 is according to embodiment 7, wherein the immobilized enzymes comprise choline oxidase, glucose oxidase, or combinations thereof.

[0013] An embodiment 9 is according to any of embodiments 1-8, wherein the signal zone is provided by a porous substrate that is in direct contact with the working electrode and the signal zone and the working electrode are in fluid communication.

[0014] An embodiment 10 is according to any of embodiments 1-9, wherein the detection zone and the signal zone are provided by a porous substrate through which the fluid sample flows.

[0015] An embodiment 11 is according to embodiment 10, wherein the porous substrate comprises nitrocellulose, cotton, cellulose, glass fibers, polymer fibers, and combinations thereof.

[0016] An embodiment 12 is according to any of embodiments 1-11, further comprising a bridge zone in between the detection zone and the signal zone and in fluid communication with both.

[0017] An embodiment 13 is according to any of embodiments 1-12, wherein the working electrode comprises indium tin oxide (ITO), tin oxide, zinc oxide, a metal oxide semiconductor, graphene, reduced graphene oxide, a conductive polymer, or a conductive nanostructured material.

[0018] An embodiment 14 is according to any of embodiments 1-13, wherein the fieldeffect transistor is not in fluid communication with the detection zone and is not in fluid communication with the signal zone.

[0019] An embodiment 15 is according to any of embodiments 1-14. wherein the working electrode is in electrical communication with the gate of the field-effect transistor and the reference electrode is configured to apply a gate voltage to the field-effect transistor.

[0020] An embodiment 16 is according to any of embodiments 1-15, further comprising a microcontroller unit in electrical communication with the field-effect transistor and configured to apply a drain voltage to the field-effect transistor and further configured to apply a gate voltage to the field-effect transistor via the reference electrode.

[0021] An embodiment 17 is according to any of embodiments 1-16, further comprising an operational amplifier assembly in electrical communication with the field-effect transistor and configured to measure an output voltage corresponding to the change in surface potential of the working electrode.

[0022] An embodiment 18 is according to any of embodiments 1 -17, further comprising a display configured to display an output voltage corresponding to the change in surface potential of the working electrode or a concentration value corresponding to a concentration of the biomarker in a test specimen or both, wherein the concentration value is determined from the output voltage.

[0023] An embodiment 19 is according to any of embodiments 1-18. wherein the sample unit and the measuring unit and configured to detach from one another; one or both of the sample unit and the measuring unit are configured to be held by a hand of a user; or both.

[0024] An embodiment 20 is a method for electrically detecting a biomarker, the method comprising: flowing the fluid sample through the detection zone of the system of claim 1, the fluid sample comprising an enzyme, a radical substrate, a primary antibody for the biomarker, and a test specimen, wherein at least some biomarkers in the fluid sample are captured in the detection zone; flowing uncaptured components of the fluid sample to the signal zone, wherein reactions between uncaptured enzymes and uncaptured radical substrates generate protons; and measuring the change in surface potential of the working electrode due to generated protons using the field-effect transistor of the measuring unit.

[0025] An embodiment 21 is according to embodiment 20, wherein the enzyme is a component of a biomarker-enzyme conjugate comprising the biomarker and the enzyme.

[0026] An embodiment 22 is according to embodiment 21, wherein the biomarkerenzyme conjugate further comprises a nanoparticle having a surface on which multiple biomarker-enzyme complexes are bound.

[0027] An embodiment 23 is according to any of embodiments 20-22, further comprising forming the fluid sample by combining the enzy me, the radical substrate, the primary' antibody for the biomarker, and the test specimen and incubating for an incubation time.

[0028] An embodiment 24 is according to any of embodiments 20-23, wherein the enzyme catalyzes oxidation of the radical substrate using a peroxide.

[0029] An embodiment 25 is according to any of embodiments 20-24, wherein the radical substrate is a phenolic compound.

[0030] An embodiment 26 is according to embodiment 25, wherein the phenolic compound is selected from fluorophenol, 4-iodophenol. 4-chlorophenol, 4-bromophenol. and combinations thereof.

[0031] An embodiment 27 is according to any of embodiments 20-26, wherein the fluid sample further comprises a reagent used by an enzyme to produce a peroxide.

[0032] An embodiment 28 is according to embodiment 27, wherein the reagent is choline chloride or glucose.

[0033] Other principal features and advantages of the disclosure will become apparent to those skilled in the art upon review of the following drawings, the detailed description, and the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Illustrative embodiments of the disclosure will hereafter be described with reference to the accompanying drawings.

[0035] FIG. 1 A shows images of an illustrative system according to the present disclosure that includes a handheld device and cartridge. FIG. IB is a schematic diagram illustrating the operational w orkflow'. The signal generation mechanisms by HRP and substrate reaction are shown for (FIG. 1C) conventional optical systems, (FIG. ID) the REEA system, and (FIG. IE) proton detection as a result of the REEA reaction via FET.

[0036] FIG. 2A show s the chemical structures of the substrates tested for the REEA system. FIG. 2B shows the distribution of Vth versus substrate reacted with HRP and H2O2, including controls with substrate alone, substrate with H2O2 but no HRP. and substrate wdth HRP but no H2O2. FIG. 2C plots detection signals as a function of substrate type. FIG. 2D plots changes in AH and AG simulated by DFT for each substrate type. FIG. 2E plots Vth versus HRP concentration at 10 mM FP and 5 mM H2O2 diluted in either PBS or plasma.

[0037] FIG. 3A show s signal comparison from paper fluidics strips spotted w ith anti-E2 antibodies (0 to 100 pg / mL), analyzed using the REEA system and the handheld device, alongside colorimetry. The inset shows images of paper fluidics strips characterized using TMB. FIG. 3B shows a schematic of the automated H2O2 generation system, illustrating theinteraction between ChOx and CC and the subsequent HRP-FP reaction. FIG. 3C plots AVout of paper fluidics dried with ChOx and analyzed with AuNP-HRP-E2. CC, and each REEA substrate. The inset shows components of the cartridge and the testing solution. FIG. 3D plots AVout of paper fluidics dried with vary ing concentrations of ChOx (for testing solution: AuNP-HRP-E2 at 0.05 OD, CC at 25 mM, and FP at 5 mM). FIG. 3E plots AVout of paper fluidics dried with 10 mg / mL ChOx in response to different CC concentrations (for testing solution: AuNP-HRP-E2 at 0.05 OD, and FP at 5 mM). FIG. 3F plots AVout of paper fluidics dried with 10 mg / mL ChOx in response to varying FP concentrations (for testing solution: AuNP-HRP-E2 at 0.05 OD, and CC at 25 mM).

[0038] FIGS. 4A-4B plots capture efficiency, signal resolution, and proton generation efficiency with (FIG. 4A) each surfactant and (FIG. 4B) stabilizer mixed into a 2% BSA solution.

[0039] FIG. 5 A show s a schematic of REEA cartridge operation with incubation method.FIG. 5B plots SNR as a function of AuNP-HRP-E2 conjugate concentration in testing samples. FIG. 5C plots AVout and CV values in response to spiked E2 concentrations ranging from 328 fg / mL to 1 ng / mL. FIG. 5D shows a comparison of E2 testing results obtained from the REEA system and an FDA-cleared clinical laboratory immunoassay (Cobas e801, n=23).

[0040] FIG. 6 is a schematic of an illustrative system according to the present disclosure including the handheld device and cartridge. The handheld device is compact, with dimensions of approximately 2.5 cm by 4 cm and features a display screen for real-time readouts. The paper fluidics cartridge is designed to facilitate sample movement and reaction processes. A reference electrode (a copper electrode) is used to apply gate voltage (VG) for the FET from the microcontroller unit (MCU) integrated into the handheld device. The drain voltage (VD) is independently supplied to the drain terminal by the MCU. The ITO working electrode built in the paper fluidics cartridge is remotely connected to the gate of the FET built into the handheld device. Once the testing sample is injected into the main chamber, the electrical connection betw een the working and reference electrodes is established. The output voltage in the handheld device system is derived from the voltage drop across the resistance at the drain terminal, measured through differential voltage amplification using an operational amplifier (op-amp). The output voltage is closely related to the drain current, essentially representing the drain current multiplied by a constant resistance. In other w ords, a shift in the threshold voltage results in a change in the drain current observed in the transfer curves ofFET, which, in turn, results in a corresponding change in the output voltage (Vout) of the handheld device.DETAILED DESCRIPTION

[0041] In one aspect, systems for electrically detecting a biomarker are provided. The system comprises a sample unit comprising a sample chamber configured to contain a fluid sample; a reference electrode in fluid communication with the sample chamber; a detection zone in fluid communication with the sample chamber and configured to capture a biomarker; a signal zone in fluid communication with the detection zone; and a working electrode configured such that its surface potential changes in the presence of charged species (e g., protons, electrons, charged elements or molecules) generated in the signal zone (e g., due to a reaction occurring in the signal zone such as that between an enzyme and a radical substrate that generates the charged species). The sy stem further comprises a measuring unit comprising a field-effect transistor (FET) in electrical communication with the reference electrode and the working electrode of the sample unit, the measuring unit configured to measure the change in surface potential of the working electrode due to the generated charged species. The measured surface potential change provides an indication of the presence (or absence) of the biomarker in the fluid sample and may further provide an amount of the biomarker in the fluid sample.

[0042] By "‘fluid communication” it is meant that when a fluid sample is present in the system (as during its operation), the fluid sample is present, and / or can flow, between components that are in fluid communication with one another. By “electrical communication” it is meant that during operation of the system, carriers (e.g., electrons) can flow between components that are in electrical communication with one another.

[0043] An image of an illustrative system 100 is shown in FIG. 1A. A schematic of system 100 is shown in greater detail in FIG. 6. The system 100 comprises a sample unit 102 (which may be referred to herein as a cartridge) and a measuring unit 104 (which may be referred to herein as a handheld device). In FIG. 6, the sample unit 102 and the measuring unit 104 are shown mounted to one another. This mounting may be detachable as schematically shown in FIG. IB, depicting the FET 120 of the measuring unit 104 in the process of being attached to the sample unit 102. The shape and dimensions of one or both of the measuring unit 104 and the sample unit 102 may be such that they can be held by a hand of a user of the system 100.

[0044] Referring back to FIG. 6, the sample unit 102 is configured to contain the fluid sample as well as to allow the fluid sample (or components thereof) to flow between components of the sample unit 102 so that it can be processed as further described below. The sample unit 102 comprises a sample chamber 106 configured to contain the fluid sample. For example, the sample chamber 106 may be provided by a well or depression defined in a base 108. The sample unit 102 further comprises a reference electrode 110 (e.g., a copper electrode) mounted and positioned on the base 108 such that it is in fluid communication with the sample chamber 106. The sample unit 102 further comprises a detection zone 112 also mounted and positioned on the base 108 such that it is in fluid communication with the sample chamber 106. Thus, when a fluid sample is present in the sample chamber 106, the fluid sample is in contact with both the reference electrode 110 and the detection zone 112. The reference electrode 110 may be in direct contact with the detection zone 112, but need not be, as shown in FIG. 6.

[0045] Although not show n in FIG. 6, the detection zone 112 is configured to capture a biomarker present in the fluid sample. This may be accomplished by the detection zone 112 comprising immobilized secondary' antibodies capable of capturing a primary antibody of the biomarker. The immobilization may be achieved using known techniques for binding biomolecules to material surfaces, including the material from which the detection zone 112 is composed. (See also the techniques used in the Example, below.) Selection of the secondary antibodies may depend upon the desired biomarker to be detected. In embodiments, the biomarker is a fertility’ hormone such as estradiol (E2) and the primaryantibody is an E2 antibody. (See FIG. 5B.) Other biomarkers that may be used include progesterone and luteinizing hormone. In general, biomarkers that may be detected and / or quantified by the present systems include hormones, proteins, chemicals, biological agents, viruses, and bacteria.

[0046] Referring back to FIG. 6, the sample unit 102 further comprises a signal zone 114 in fluid communication yvith the detection zone 112. Such fluid communication may be accomplished by a bridge zone mounted and positioned relative to both the signal zone 1 14 and the detection zone 112 such that these three components are in fluid communication -with one another. In this w ay, a fluid sample (or components thereof) flowing through the detection zone 112 can further flo v through the bridge zone and then, the signal zone 114. The vertical arrow on the base 108 shows the direction of fluid flow which shows that the bridge zone may be downstream from the detection zone 112 and the signal zone 1 14, may bedow nstream from both the bridge zone and the detection zone 112. Each of these zones may be arranged in a planar configuration relative to one another, i.e., arranged on the same plane (versus a vertical, stacked configuration). As shown in FIG. 6, although the signal zone 114 is in fluid communication with the detection zone 112 via the bridge zone, the signal zone 114 need not be (and generally is not) mounted and positioned in contact with the sample chamber.

[0047] The detection zone 112, the signal zone 114. and the bridge zone may be provided by pieces of a porous substrate mounted to the base 108. Each zone may constitute a distinct region on or within a piece of porous substrate or an entire piece of porous substrate may provide a respective zone. Various materials may be used for the porous substrate, e.g., nitrocellulose, cotton, cellulose, glass fibers, polymer fibers, or combinations thereof. For example, the detection zone 112 may be provided by a first strip of nitrocellulose mounted to the base 108 and the signal zone 114 may be provided by a second strip of nitrocellulose also mounted to the base 108, but separated from the detection zone 112 by a gap 116. The bridge zone may be provided by a piece of cotton mounted in the gap 116 such that it forms a bridge through which the fluid sample may flow from the detection zone 112 to the signal zone 114. This configuration provides flexibility such that the various zones may be configured differently from one another, while still allowing the fluid sample to move therethrough. For example, the bridge zone and the signal zone 114 need not comprise (and generally do not comprise) the immobilized secondary antibodies of the detection zone 112.

[0048] Although not shown in FIG. 6, due to the flexibility' in the design as described above, the signal zone 114 may be configured to induce the reaction betw een the enzyme and the radical substrate occurring in the signal zone 114 that generates the protons. (As noted above, other reactions that generate other charged species are encompassed.) This may be accomplished by the signal zone 114 comprising an immobilized enzyme that produces a chemical compound required for the reaction betw een the enzyme and the radical substrate. The immobilized enzyme in the signal zone 114 and the enzyme that reacts with the radical substrate to generate the protons in the signal zone 114 are generally not the same enzy me. Selection of the immobilized enzyme in the signal zone 1 14 depends upon the required chemical compound and thus, upon the enzyme and the radical substrate undergoing the proton generating reaction. In embodiments, this reaction involves an enzyme that is capable of catalyzing oxidation of the radical substrate using a peroxide (e.g.. H2O2), i.e.. a peroxidase, such as horseradish peroxidase (HRP). Thus, the reaction between the enzymeand the radical substrate occurring in the signal zone 114 may refer to the oxidation of the radical substrate as catalyzed by the enzyme using the peroxide. (See FIG. ID, wherein HRP is the enzyme and PhOH refers to the radical substrate.) The phrase “radical substrate” refers to a chemical species (e.g., chemical compound) that is converted to a radical and releases a proton upon oxidation as catalyzed by the enzy me. The radical substrate may be a phenolic compound comprising a phenol moiety, e.g., fluorophenol, 4-iodophenol. 4-chlorophenol, 4- bromophenol, and combinations thereof. (See FIG. 2A.) In embodiments, the immobilized enzyme in the signal zone 114 is one capable of producing the peroxide, such as choline oxidase (ChOx). (See top image of FIG. 3B.) Other immobilized enzymes that may be used include glucose oxidase. The amount of immobilized enzy me in the signal zone 114 may be optimized as described in the Example, below. (See FIG. 3D.) The immobilization may be achieved by known techniques for binding biomolecules to material surfaces, including any of the porous substrates described above. The detection zone 112 and the bridge zone need not comprise (and generally do not comprise) the immobilized enzy mes of the signal zone 114. As such, each of the detection zone 112, the bridge zone, and the signal zone 114 are distinctly configured regions in the present systems with different physical structures and thus, different functions.As shown in FIG. 6, the sample unit 102 may comprise a single detection zone 112, a single signal zone 114, and if present, a single bridge zone. However, more than one of each type of zone may be used (e.g., multiple detection zones). Similarly, other zones with other configurations may be included in the sample unit 102, depending upon the type of processing to be carried out on the fluid sample.

[0049] Referring back to FIG. 6. the sample unit 102 further comprises a working electrode 1 18 configured such that its surface potential changes in the presence of protons generated by the reaction between the enzyme and the radical substrate occurring in the signal zone 114. (As noted above, other reactions that generate other charged species are encompassed.) This may be achieved by forming the working electrode 118 from an ion- selective electrode on which protons can bind (e.g., a metal oxides such as tin oxide, indium tin oxide, zinc oxide, and other metal oxide semiconductors; graphene; reduced graphene oxide; a conductive polymer; a conductive nanostructured (e.g., two-dimensional nanostructured) material), resulting in a change in the surface potential thereof, and mounting and positioning the working electrode 118 on the base 108 such that it is in fluid communication with the signal zone 114. As shown in FIG. 6, the working electrode 118 maybe in direct contact with the signal zone 114, i.e., a surface of the working electrode 118 may contact a surface of the signal zone 114 such that an interface is formed therebetween, which means there is no space or gap present between respective surfaces. In other embodiments, the working electrode is in direct contact with a porous substrate on or in which the signal zone 114 is formed as a distinct region thereof. In such an embodiment, an interface is formed between the working electrode 118 and the porous substrate with no space or gap so that the working electrode 118 and the signal zone 114 are in fluid communication with one another. The mechanism of proton generation, binding, and detection is schematically illustrated in FIG. IE.

[0050] The system 100 further comprises a measuring unit 104 configured to measure the change in surface potential of the working electrode 118 due to the protons generated in the signal zone 114. (As noted above, other charged species are encompassed.) The measuring unit 104 comprises a field-effect transistor (FET) 120 comprising a source, a drain, and a gate, and in electrical communication with the reference electrode 110 and the working electrode 118 of the sample unit 102. Although the FET 120 is in electrical communication with components of the sample unit 102, it is not in fluid communication with the sample unit 102. This includes the FET 120 not being in fluid communication with the detection zone 1 12, the signal zone 114, the reference electrode 110, or the working electrode 118. Commercially available metal-oxide semiconductor FETs (MOSFETs) may be used.Regarding the electrical communication between the sample unit 102 and the measuring unit 104, as shown in FIG. 6, the working electrode 118 may be in electrical communication with the gate of the FET 120. The reference electrode 110 may be configured to apply a gate voltage (VG) to the FET 120, e.g., via a microcontroller unit (MCU) 122 also in electrical communication with the FET 120. The MCU 122 may be further configured to apply a drain voltage (VD) the FET 120. Commercially available MCUs may be used.

[0051] Measurement of the change in surface potential of the working electrode 118 may be facilitated by using an operational amplifier (op-amp) assembly 124 also in electrical communication with the FET 120. For example, in the system 100, the op-amp assembly 124 is configured to measure an output voltage (Vout) as a voltage drop across a fixed resistor 126 at the drain of the FET 120 measured through differential voltage amplification using an opamp 128 in the op-amp assembly 124. Vout is proportional to the drain current of the FET 120 which, in turn, corresponds to the change in surface potential of the working electrode 118. The measuring unit 104 may further include a display 130 in electrical communication withthe op-amp assembly 124 and configured to display Vout, including as a function of time (see panel 4 of FIG. IB).

[0052] It is understood that system 100 shown in FIG. 6 is illustrative and systems for electrically detecting biomarkers may include additional, fewer, different components, and / or different arrangements as compared to those shown in FIG. 6. For example, an additional controller may be included (or the MCU 122 appropriately configured) that comprises a processor and an application that performs other various operations. Such operations include receiving and / or processing data, e.g., converting Vout to an associated concentration value of the biomarker in the fluid sample. This may involve comparison to a calibration plot stored in, or operably coupled to, the controller. As another example, other electronic circuitry or assemblies different from those shown in FIG. 6 may be electrically coupled to the FET to measure any electrical characteristics of the FET that change due to the change in surface potential, e.g., threshold voltage (Vth). drain current, etc.

[0053] In another aspect, methods for electrically detecting a biomarker using any of the disclosed systems, including system 100, are also provided. Using system 100 as an illustrative embodiment, such a method comprises flowing a fluid sample through the detection zone 112. This may occur spontaneously after the fluid sample is injected into the sample chamber 106 by a user, i.e., through capillary action by the fluid sample being drawn up by the porous substrate from which the detection zone 112 is composed. This may be facilitated by use of an absorbent pad as show n in FIG. 6.

[0054] The fluid sample comprises the enzyme (e.g., HRP), the radical substrate (e.g., any of the disclosed phenolic compounds), the primary antibody for the biomarker (e.g., E2), and a test specimen. The enzyme of the fluid sample is generally provided as a component of a biomarker-enzyme conjugate comprising the biomarker and the enzyme. The biomarkerenzyme conjugate may further comprise a nanoparticle (e.g., a gold nanoparticle) having a surface on which multiple biomarker-enzyme complexes are bound. An illustrative such conjugate is schematically shown and labeled AuNP-HRP-E2 in panel 1 of FIG. 5 A (see also panel 2 of FIG. 3B). Known techniques may be used to form such conjugates. (See also the techniques used in the Example, below.) The test specimen may be a fluid (e.g., blood. serum, plasma, saliva, urine, sweat, etc.) from a mammalian subject (e.g., a human). The fluid sample comprises other components, e.g., one or more of a solvent, a surfactant, and a stabilizer. Illustrative solvents, surfactants, and stabilizers are provided in the Example,below. If the signal zone 114 comprises immobilized enzy mes (e.g., ChOx or glucose oxidase) the fluid sample may further comprise a reagent (e.g., choline chloride, CC (for ChOx) or glucose (for glucose oxidase)) used by the immobilized enzyme to produce the chemical compound (e.g., H2O2) required for the proton generating enzyme-radical substrate reaction. Optimization of the fluid sample may be carried out as described in the Example below, e.g., for optimizing the concentration of the reagent (FIG. 3E), for optimizing the type and concentration of the radical substrate (FIGS. 2A and 3F). for optimizing the type and concentration of surfactants / stabilizers (FIGS. 4A-4B), and for optimizing the concentration of the biomarker-enzyme conjugates (FIG. 5B).

[0055] Due to the presence of the primary antibody for the biomarker in the fluid sample, biomarkers present in the test specimen and / or biomarkers in the biomarker-enzyme conjugates bind to the primary antibody. However, this binding is competitive due to the differential binding affinities of test specimen biomarkers versus biomarkers in the biomarker-enzyme conjugates. Specifically, test specimen biomarkers have a higher binding affinity' to the primary antibodies as compared to biomarkers in the biomarker-enzyme conjugates. Thus, the greater the amount of biomarkers present in the test specimen, the greater the amount will be bound to the primary antibodies relative to the biomarkers in the biomarker-enzyme conjugates. Prior to flowing the fluid sample through the detection zone 112, the method may comprise incubating the fluid sample for a period of time (incubation time) to facilitate biomarker binding to the primary antibodies. The incubation time may be optimized as described in the Example, below.

[0056] While flowing the fluid sample through the detection zone 112, at least some biomarkers (bound to the primary antibodies) in the fluid sample are captured, e.g., via the immobilized secondary antibodies. The captured biomarkers may be those present in the test specimen and / or those in the biomarker-enzyme conjugates. However, as noted above, the greater the amount of test specimen biomarkers, the greater the amount will be bound to the primary antibodies, resulting in a greater amount of captured test specimen biomarkers relative to the captured biomarker-enzyme conjugates. This, in turn, results in a greater amount of uncaptured biomarker-enzyme conjugates.

[0057] Next, the method comprises flowing uncaptured components of the fluid sample through the signal zone 114. Again, this may occur spontaneously as the uncaptured components are drawn up by the porous substrate from which the signal zone 114 iscomposed (including via the bridge zone). The uncaptured components may include uncaptured biomarker-enzyme conjugates as well as the radical substrate and the reagent. As noted above, due to differential binding affinities, a greater amount of test specimen biomarkers captured in the detection zone 112 results in a greater amount of uncaptured biomarker-enzyme conjugates flowing through the signal zone 114. In the signal zone 114, reactions between the enzymes of uncaptured biomarker-enzyme conjugates and radical substrates generate protons. As described above, these reactions may be induced by immobilized enzymes (e g., ChOx) in the signal zone 114 that produce the chemical compound (e.g., H2O2) using the reagent (e.g., CC) also present in uncaptured components of the fluid sample. As also described above, these generated protons bind to the surface of the working electrode 118, resulting in a change in its surface potential. A greater amount of uncaptured biomarker-enzyme conjugates results in a greater amount of generated protons and thus, a greater positive change in the surface potential of the working electrode 118.

[0058] Next, the method comprises measuring the change in surface potential of the working electrode 118 using the measuring unit 104. As described above, this may be accomplished by the working electrode 118 being in electrical communication with the gate of the FET 120 while the reference electrode 110 applies the gate voltage to the FET 120 and the MCU 122 applies the drain voltage to the FET 120. A greater positive change in surface potential due to a greater amount of generated protons reduces a threshold voltage (Vth) of the FET 120, thereby increasing the drain current of the FET 120. As also described above, the measured change in surface potential may be provided as a Vout, measured by the op-amp assembly 124, which is directly proportional to the drain current of the FET 120. A greater drain current results in a greater Vout. As also described above, the Vout may be further converted to a concentration value of the biomarker. A greater Vout corresponds to a greater concentration of the biomarker present in the test specimen.

[0059] The present methods are further illustrated by reference to FIG. 5A and the system 100 shown in FIG. 6. First (panel 1), a fluid sample is prepared by combining a biomarker- enzyme-nanoparticle conjugate (AuNP-HRP-E2), a radical substrate (fluorophenol, FP), a reagent (choline chloride, CC), a primary antibody (E2 antibody), and a test specimen (serum containing an unknown amount of E2). Next (panel 2), the fluid sample is incubated for an incubation time to allow the biomarkers in the test specimen and in the biomarker-enzyme- nanoparticle conjugates to competitively bind to the primary antibodies. After injection into the sample chamber 106 of the sample unit 102, as shown in panel 3, the fluid sample flow sthrough the detection zone 112, wherein at least some biomarkers in the fluid sample (bound to the primary antibodies) are captured by immobilized secondary’ antibodies. Next (panel 4), uncaptured components of the fluid sample (unbound biomarker-enzyme-nanoparticle conjugates, FP, and CC) flow through the bridge zone. Next (panel 5), these uncaptured components flow through the signal zone 114, wherein reactions between immobilized enzyme (ChOx) and the reagent (CC) to produce H2O2. The H2O2 induces reactions between enzymes (HRP) of the unbound biomarker-enzyme-nanoparticle conjugates and radical substrates (FP) to generate protons which bind to the working electrode 118 (ITO), thereby changing its surface potential. As shown in panel 6, the change in surface potential is measured using the FET 120 of the measuring unit 104.

[0060] The methods above have been illustrated by reference to an enzy me (e.g., HRP) reacting with a radical substrate (e.g., a phenolic compound) to generate protons in the signal zone 114. However, as noted above, other reactions that generate other charged species in the signal zone 114 are encompassed. FIG. As another example, HRP can react with 3, 3', 5,5'- Tetramethylbenzidine (TMB) to generate charged species in the signal zone 114.Specifically, TMB is oxidized by HRP to form a charge-transfer complex, specifically a radical, which in turn produces the signal in the present systems.EXAMPLE

[0061] INTRODUCTION

[0062] This Example presents a diagnostic platform that integrates REEA with a paperbased fluidic system and a handheld FET reader. The FET quantifies protons generated within the paper fluidics cartridge by the REEA system, correlating the proton count with the concentration of target analytes. As a proof-of-concept, estradiol (E2), a key fertility hormone, w as measured with a limit of detection (LOD) of 146 fg / mL under E2-spiked buffer condition and a coefficient of variation below 9.2%, using a cartridge costing $0.55 per test. The system demonstrated a strong correlation (r2= 0.963) across a measuring range of 19 to 4,551 pg / mL for 23 plasma samples, with a standard error of estimate (Sy / x) of 0. 15 in a logarithmic regression analysis. These results are comparable to those obtained with an FDA-cleared clinical immunoassay on the Cobas e801 analyzer, indicating high accuracy. Screening 14 phenolic compounds interacting with HRP identified halogenated phenolic substrates — such as 4-fluorophenol (FP), 4-iodophenol (IP), 4-chlorophenol (CP), and 4- bromophenol (BP) — as the most effective for REEA. Additionally, the REEA cartridge w asequipped with an enzymatic choline oxidase (ChOx)-based system for automated hydrogen peroxide (H2O2) generation. The medical significance of this technology lies in its abi 1 i ty to enable early detection and monitoring of various health conditions, including hormonal imbalances, metabolic disorders, chronic diseases, and infectious diseases. This system's capacity for timely and accurate diagnostics will enhance patient outcomes and reduce healthcare costs.

[0063] METHODS

[0064] Chemicals and Other Resources. The following chemicals were purchased for the study: 2,4,6-Trimethoxyphenol (TMYP) (Sigma Aldrich, AMBH303C58D3), 2,4,6- Trimethylphenol (TMLP) (Sigma Aldrich, T79006-25G). 4-Methoxy phenol (MP) (Sigma Aldrich, M18655), 4-fluorophenol (FP) (Sigma Aldrich, F13207), 4-iodophenol (IP) (Sigma Aldrich, 110201), 4-chlorophenol (CP) (ThermoFisher Scientific, 181001000), 4- bromophenol (BP) (Sigma Aldrich, B75808), 2,4,6-trichlorophenol (TCP) (Sigma Aldrich, T55301). p-anisidine (AD) (Sigma Aldrich, A88255). p-toluidine (T) (Sigma Aldrich, 236314), 4-fluoroaniline (FA) (Sigma Aldrich, F3800), o-phenylenediamine (OPD) (Sigma Aldrich, P23938), 2,4,6-trimethylxyanilline (TMYA) (Sigma Aldrich, AMBH303C5E71), and 4-chloroaniline (CA) (Sigma Aldrich, C22415). Additionally, anti-17 beta-estradiol monoclonal antibody (CD Creative Diagnostics, DMATBT-49063MH), E2-HRP conjugate (CD Creative Diagnostics. DAGA-025B-HRP). and 17-beta-estradiol standard (CD Creative Diagnostics, DAGS030) were acquired. 3,3',5,5'-tetramethylbenzidine dihydrich (TMB) (Sigma Aldrich, 87750), choline chloride (CC) (Sigma Aldrich, C7527-100G), horseradish peroxidase (HRP) (Toyobo, PEO-301), and choline oxidase (ChOx) (Toyobo, CHO-301) were also obtained. Indium-tin-oxide on polyethylene terephthalate (ITO / PET) (Sigma Aldrich, 639303) was used as the sensing electrode. Gold nanoparticles (Ted Pella, 15704-1), acetonitrile (Sigma Aldrich, 34851), 10% BSA block (Thermo Scientific, 37525), mouse IgG-HRP (SouthemBiotech, 0107-05), goat anti-mouse IgG (SouthemBiotech, 1033-01), goat anti -mouse IgG-HRP (SouthemBiotech, 1033-05), hydrogen peroxide (H2O2) solution (Sigma Aldrich, 88597), and estradiol (Sigma Aldrich, El 024) were purchased for the study.

[0065] Solution Test. ITO / PET was cut into 1 x 2 cm2pieces and cleaned with isopropanol for 20 minutes before use in a standard remote-gate FET (RGFET) setup with an Ag / AgCl reference electrode.

[0066] For the solution-based tests, the optimal FP concentration for the REEA was determined to be 10 mM, as higher concentrations resulted in signal saturation. Therefore, a concentration of 10 mM was used in all subsequent experiments to ensure consistency and accuracy in the REEA process during solution testing.

[0067] For FIG. 2B, IM of each phenolic substrate was initially diluted in acetonitrile. Each substrate solution and H2O2 were further diluted in lx PBS to final concentrations of 30 mM and 15 mM, respectively. HRP was also diluted to 3 pg / mL in lx PBS. Sequentially, 10 pL of each solution was added to the ITO electrode in the following order: HRP solution, substrate solution, and H2O2 solution, resulting in final concentrations of 10 mM for the substrate, 5 mM for H2O2, and 1 pg / mL for HRP, with a total testing volume of 30 pL. For FIG. 2E, different concentrations of HRP, ranging from 1 pg / mL to 100 pg / mL, were prepared in lx PBS and human plasma (purchased from Kalen Biomedical). An Ag / AgCl reference electrode was immersed in the solution during measurements. Each ITO electrode was discarded after a single measurement, and the standard Ag / AgCl reference electrode was washed in 70% ethanol for 5 minutes, followed by rinsing with DI water three times before being used in the next experiment.

[0068] For the solution-based tests using the ITO-Cu electrode, the ITO electrode was laser-cut into a spoon shape using an OMTech laser cutter. The cutting was performed at a speed of 50 mm / s with a current of 0.25 A. The circular part of the spoon-shaped electrode had a diameter of 6 mm, and a 2 mm x 2 mm notch w as cut to accommodate a rectangular Cu electrode (2 mm x 20 mm), which was inserted into the notch.

[0069] For the solution tests, 10 pL of HRP solution, diluted in IX PBS to different concentrations, was first placed on the electrode. Subsequently, 10 pL of 30 mM FP solution (diluted in acetonitrile) and 10 pL of 15 mM H2O2 solution were sequentially added, resulting in final concentrations of 5 mM H2O2 and 10 mM FP in the test solution. Each ITO-Cu electrode was discarded after a single measurement.

[0070] Cartridge Fabrication. Following the cleaning process, ITO (1 x 3 cm2) and Cu (1 x 3 cm2) electrodes w ere laser-cut to the desired dimensions using an OMTech laser cutter at a speed of 50 mm / s and a current of 0.25 A. Paper fluidic strips were constructed using a nitrocellulose (NC) membrane (MilliporeSigma, HF13502XSS) mounted on laminated cards (MilliporeSigma, HF000MC100) with release liners. The NC strip in the signal zone w as spotted with 10 pL of 10 mg / mL ChOx diluted in 1 x PBS, which w as determined to be theoptimal concentration per FIG. 3D. The detection zone was spotted with 5 pL of 200 pg / mL goat anti-mouse IgG in 0.2% BSA solution to ensure sufficient antibody concentration for capturing 1.5 pg / mL E2 antibody in the running buffer. NC strips were dried for 15 minutes at 37 °C and stored with silica gel. Each NC strip was affixed to the designated areas of the cartridge's acrylic frame. To provide a bridge zone, a cotton linter pad (CF7, Cytiva), sliced into 0.4x0.6 cm, was placed between the detection and signal zones using an acrylic jig supported by a clip. The ITO was mounted over the ChOx-functionalized NC strip in the signal zone using a magnet.

[0071] Assay Operation. For colorimetry tests in FIG. 3A, the NC strip functionalized with 2.5 pL of anti- 17 beta-estradiol monoclonal antibody diluted in 0.1% BSA was affixed onto the cartridge acryl flame and loaded 100 pL of 1 pg / mL anti -mouse IgG-HRP in 2% BSA in PBS solution for 5 minutes. Then, 200 pL of 2% BSA washing buffer was injected for 3 minutes through the main chamber of cartridge, followed by 100 pL of 20% TMB solution. New NC strips from the same batch underwent the same washing step and were characterized by injecting 100 pL of 5 mM FP and 5 mM H2O2 solutions.

[0072] All NC strips used in FIGS. 3C-3F were functionalized with 10 pL of 10 mg / mL ChOx diluted in l x PBS. 15-nm-sized AuNPs were used for E2-HRP conjugation. AuNP- HRP-E2 conjugates were synthesized by adding 1 mL of AuNP solution (1 OD), 100 pL of 0. 1 M borate buffer (pH 8.5), and 10 pL of 1 mg / mL E2-HRP into a sterile Eppendorf tube. The mixture was incubated for 45 minutes at room temperature, followed by the addition of 100 pL of 1% BSA in PBS to act as a blocking agent, preventing nonspecific binding. After a 30-minute incubation, the AuNP conjugates were centrifuged at 4°C for 15 minutes at 15,000 rpm, washed three times with 1 mL of 10 mM Tris buffer (pH 7.4). and resuspended in 100 pL of storage buffer (0. 1 M borate buffer, pH 8.5, with 0. 1% BSA and 1% sucrose). The final concentration of AuNP-E2-HRP conjugates was confirmed by OD measurement at 525 nm using a Biotek PowerWave XS Microplate Reader.

[0073] For testing solutions used in FIG. 3C, IM substrates were diluted in acetonitrile, then further diluted in 2% BSA to achieve a final concentration of 5 mM which is the optimized concentration as shown in FIG. 3F. Similarly, IM CC was diluted in de-ionized water and then in 2% BSA to a final concentration of 25 mM which is the optimized concentration shown in FIG. 3E. AuNPs were added to the testing solution, resulting in a final concentration of 0.05 OD. A total of 200 pL of the testing solution, containing differentsubstrates at the same concentration (5 mM), was injected into the NC strips for FIG. 3C through main chamber of the cartridge flame. For FIG. 3D, NC strips w ith dried ChOx concentrations ranging from 0 to 11 mg / mL were prepared. A total of 200 pL of testing solution, containing 0.05 OD AuNP-HRP-E2 conjugate, 25 mM CC, and 5 mM FP, was used, with 2% BSA in 1 x PBS as the base buffer.

[0074] In FIGS. 3E and 3F, a total of 200 pL testing solution for FIG. 3E included vary ing concentrations of CC, ranging from 0.048 mM to 100 mM, while maintaining 0.05 OD AuNP-HRP-E2 conjugate and 5 mM FP with 2% BSA in lx PBS as the base buffer. For FIG. 3F, a total of 200 pL testing solution contained varying concentrations of FP, ranging from 0.015 mM to 10 mM, while maintaining 0.05 OD AuNP-HRP-E2 conjugate and 25 mM CC with 2% BSA in 1 x PBS as the base buffer.

[0075] The optimized running buffer through experiments in FIGS. 4A and 4B contained 2% BSA. 0.2% IGEPAL, and 2.5% PVP. The running buffer including 2% BSA. 0.2% IGEPAL, and 2.5% PVP was further mixed with either E2-spiked or clinical plasma samples for experiments in FIGS. 5B-5D. A stock solution of 17-beta-estradiol standard at a concentration of 0.5 mg / mL was prepared by dissolving the compound in isopropanol. For E2-spiked sample measurements, a 100-fold specific concentration of the E2 solution was prepared through serial dilution with isopropanol and spiked into the running buffer at a final concentration of 3% (v / v). For clinical plasma sample measurements. 5 pL of the plasma sample was mixed with 95 pL of running buffer, resulting in a final dilution factor of 60 x for operation. In both cases, 100 pL of the prepared sample solution was mixed with 100 pL of antibody solution (1.5 pg / mL E2 antibody in running buffer) and incubated for 15 minutes after vortex mixing. Then, 100 pL of substrate solution (75 mM CC, 15 mM FP in running buffer) was added, and 300 pL of this final mixture was injected into the injection zone. Measurements were taken for 10 minutes using a handheld reader.

[0076] Measurement System. All experiments for FIGS. 2B and 2E were conducted using a semiconductor analyzer with an Ag / AgCl reference electrode system. The ITO electrode of the cartridge was connected to the gate of a commercial n-type metal-oxide- semiconductor field-effect transistor (MOSFET) (CD4007UB) using an alligator clip. To ensure consistency, the same MOSFET was used throughout the experiment. The Ag / AgCl reference electrode was in contact with the solution via injections into the main chamber. Transfer curves were obtained using a Keithley 4200A semiconductor analyzer, with asource-drain voltage set at 50 mV, and a gate voltage sweep ranging from 0 to 3 V in doublesweep mode. Transfer curves were measured repeatedly for 5 minutes under each plasma sample, and Vth was calculated as the gate voltage corresponding to a drain current of 1 pA in each transfer curve. For FIGS. 3A, 3C-3F, and 5B-5C, all experiments were conducted using a handheld FET reader device and a copper reference electrode. The gate and drain voltages were fixed at 2 V and 3 V, respectively, with power supplied via a USB Type-C connection to a laptop. Vout was measured every second, and the signal was monitored in real-time through software (Arduino IDE) installed on the laptop.

[0077] Clinical Sample Test. De-identified, Lithium heparin plasma from leftover patient samples collected at The University of Chicago Medical Center with E2 concentrations ranging from 19 to 4,551 pg / mL were stored at -80 °C until use. Samples were collected under a quality assurance protocol, which qualified for an institutional review board waiver and no patient identifiers were collected. E2 concentrations were quantified using the Cobas e801 analyzer (Roche Diagnostics). After thawing, the samples were stored at 2-8 °C for up to seven days.

[0078] RESULTS

[0079] Detection Platform. The integration of FET technology with paper fluidics and the REEA system offers a low-cost, accurate, and accessible solution for at-home clinical test. FIG. 1A depicts the handheld FET reader device (dimensions 2.5 cm by 4 cm) and the paper-based immunoassay cartridge for E2 detection. The basic electrical properties of the FET used in all experiments were evaluated. The cost of the cartridge based on the materials and reagents used therein was $0.55 per test. To operate, the clinical sample is initially mixed with optimized running buffer and incubated for 15 minutes. Following incubation, the cartridge, which includes the sensing electrodes such as indium tin oxide (ITO) and reference electrodes, is electrically connected to designated terminals on the handheld device (FIG. 6). A total 300 pL of mixture of plasma and reagents is then injected into the cartridge, and the device begins real-time measurement of the signal over a 10-minute period.

[0080] REEA Mechanism. HRP, an enzy me traditionally used in analytical techniques to convert substrates into optical signals (FIG. 1C), is repurposed in the REEA system to convert aromatic substrates into radical forms, generating protons (FIG. ID). HRP. which contains a Fe(lV) oxyferryl center, undergoes oxidation by H2O2, leading to the formation of compound I (HRP-I). This compound I then interacts with a phenol compound (PhOH) at itsactive site, generating and releasing a phenolic free radical and a proton into the medium. The subsequent reaction produces compound II (HRP-II), which further reacts with another phenol molecule, releasing an additional proton. These protons, which correlate with the concentration of the target analyte, serve as the signal in the REEA system and are quantified by the FET (FIG. IE). The intrinsic pH sensitivity of the ITO electrode was measured using a remote-gate FET (RGFET) setup with a semiconductor analyzer and standard Ag / AgCl reference electrode. The transfer curved shifted as a function of pH (from 3 to 11). exhibiting parallel shifts without any change in curve shape. This behavior corresponds to a protonspecific Nemstian response of 52.4 mV / pH with an r2of 0.998, calculated based on threshold voltage (Vth) shifts across different pH levels. Additionally, no interference from variations in the surface area of the testing solution on the ITO was observed, owing to the high input impedance of the FET.

[0081] A set of phenolic and aniline compounds with a wide range of pKa values (FIG. 2A), including 2,4,6-trimethoxyphenol (TMYP), 2,4,6-trimethylphenol (TMLP), 4- methoxyphenol (MP), FP, IP, CP, BP, 2,4,6-trichlorophenol (TCP), p-anisidine (AD), p- toluidine (T). 4-fluoroaniline (FA), o-phenylenediamine (OPD), 2,4,6-trimethylaniline (TMYA), and 4-chloroaniline (CA). was screened to identify the optimal substrate for REEA in the solution phase. The screening was performed using the RGFET setup with a precise semiconductor analyzer and a standard Ag / AgCl reference electrode. Different degrees of shifts in Vth were obser ed in FIG. 2B by the reaction between HRP, H2O2, and each substrate. Without H2O2 and HRP. and with either H2O2 or HRP alone, changes in Vth were minimal for all substrates.

[0082] The detection signal, defined as the difference in Vth between the HRP / H2O2 / substrate mixture and the substrate-only condition, revealed that halogenated phenolic compounds — specifically FP, IP, CP, and BP — generated the most substantial signals (FIG. 2C). The oxidation of these compounds results in the release of halogenated ions, leading to the formation of hydrofluoric acid (HF), hydroiodic acid (HI), hydrochloric acid (HC1), and hydrobromic acid (HBr), respectively (inset of FIG. 2B). These strong acids contribute to a significant reduction in Vth. Density functional theory (DFT) simulations conducted to calculate changes in enthalpy (AH) and Gibbs free energy' (AG) supports that proton generation is highly favorable for these halogenated ions (FIG. 2D). FP was selected for subsequent experiments due to its high sensitivity, relative safety, commercial availability, and good solubility’. Compared to IP, CP, and BP, FP is easier to handle with lower toxicityand demonstrates greater applicability in practical experimental settings. These combined advantages made FP the most appropriate choice for optimizing the REEA platform.

[0083] The REEA system effectively addresses Debye length issues, as demonstrated by significant Vth shift in both IX phosphate-buffered saline (PBS) and plasma with HRP concentrations ranging from 10 pg / mL to 1 pg / mL (FIG. 2E). The transfer curves exhibited parallel shifts during the HRP-substrate reaction, without changes in shape, even in plasma samples. This indicates that the surface potential changes are specifically caused by the HRP- substrate reaction, without interference or non-specific signaling. The maximum signal window, measured from control to 1 pg / mL HRP, was determined to be approximately 0.37 V in PBS and 0.32 V in plasma. However, at HRP concentrations exceeding 10 pg / mL, a reversal in Vth was observed for both PBS and plasma, suggesting a potential saturation point in the REEA signal. This reversal is likely due to HRP inactivation, where the disruption of the heme macrocycle by phenoxyl radical attacks impairs its catalytic function.

[0084] Validation of Handheld Reader System. The real-time change in the drain current of the FET, measured at a constant gate voltage of 2 V and drain voltage of 3 V in the handheld device system, reflects alterations in surface potential on the ITO and is converted into an output voltage (Vout). Vout is derived from the voltage drop across a fixed resistor at the drain terminal, measured through differential voltage amplification using an operational amplifier (op-amp) with a 4x voltage gain. Since Vout is proportional to the drain current, it essentially represents the drain current multiplied by a constant resistance.

[0085] The real-time pH sensitivity, measured as the Vout change using the handheld FET reader with the standard Ag / AgCl reference electrode and ITO in response to the pH of the testing solution, exhibited an amplified response of 212 mV / pH, which is 4.04 times higher than the pH sensitivity measured from the Vth using the semiconductor analyzer. This enhancement is attributed to the built-in op-amp in the handheld device. The response demonstrated 99.4% linearity with negligible drift.

[0086] Potential interferences caused by various paper materials, including NC membrane, cotton linter pad, cellulous filtration paper, two glass fiber pads, and poly sulfone membrane, inserted between the ITO electrode and the solution, were found to be insignificant. This was confirmed using the handheld reader device and a standard Ag / AgCl reference electrode. That is, the Vout signal remained specific to the pH of the buffer solution, regardless of the paper type connected to the ITO electrode. The handheld FET reader'sdetection performance, as evaluated by the HRP-FP reaction in solution phase, closely matched that of a precise semiconductor analyzer.

[0087] A copper (Cu) reference electrode was used as an alternative to the standard Ag / AgCl reference electrode in the miniaturized cartridge system. The Cu reference electrode demonstrated pH sensitivity comparable to that of the Ag / AgCl electrode. Notably, the signal trend in HRP-FP reaction measured by the Cu electrode closely matched the signal from the Ag / AgCl electrode. The ITO-Cu electrode maintained a stable Vout over 5,000 s after reaching a specific value corresponding to the HRP-substrate reaction, with an insignificant drift.

[0088] Validation of REE A within paper fluidics. To demonstrate the high sensitivity of REEA system within the paper fluidic system, the LOD for both optical and REEA systems w as compared directly with a conventional lateral flow assay format. From this section onw ard, all measurements were performed using the handheld reader device and a Cu reference electrode. The NC membrane, dried with varying concentrations of anti-E2 antibody, was tested by adding anti-mouse IgG-HRP. After washing step, the strips w ere imaged using a tetramethylbenzidine (TMB) solution, producing distinct bands corresponding to different anti-E2 antibody concentrations (inset of FIG. 3A). The LOD measured by colorimetry’ was estimated to be 80 ng / mL (FIG. 3A). However, when the same paper fluidics setup w as charactenzed using the HRP-FP reaction, the LOD was determined to be at least 1,000 times lower than that achieved through colorimetry (FIG. 3A).

[0089] To enhance the HRP signal for target analyte binding, gold nanoparticles (AuNPs) were conjugated with estradiol-HRP (E2-HRP), designed for the E2 detection system. The AuNP-HRP-E2 conjugates w ere dried onto the NC membrane and tested across a concentration range of 0 to 10 optical density (OD at 525 nm). The HRP-FP reaction produced an intense signal even at a low AuNP-HRP-E2 concentration of 0.025 OD. demonstrating that AuNPs conjugated with HRP function effectively within the REEA system, as also shown in FIG. 3A.

[0090] H2O2, a key component in REEA, is unstable under ambient conditions, making automated H2O2 generation upon sample injection critical for ensuring long-term stability7and commercial scalability of the system. This was achieved through the interaction between ChOx and choline chloride (CC) in the signal zone (FIG. 6) of the cartridge. The ChOx-CC enzymatic reaction produces H2O2, which triggers the secondary enzymatic reaction betweenHRP and the REEA substrate (FIG. 3B). To validate this automated H2O2 generation system, ChOx was dried onto the NC membrane (inset of FIG. 3C) and tested with an injected solution containing AuNP-HRP-E2 conjugate (0.05 OD), 25 mM CC, and 10 mM of each substrate identified through screening (FIG. 2A). The resulting REEA signals closely matched those observed in FIG. 2B, while negligible signals were detected in negative control 1 (Nl: no substrate, with HRP and CC), negative control 2 (N2: no substrate and HRP. with CC), and the blank control (buffer only). These results confirm the system's specificity and functionality, demonstrating its effectiveness in automating H2O2 generation for the REEA process.

[0091] Using the same testing setup as shown in FIG. 3C, the concentration of ChOx dried on the NC membrane was optimized for the REEA system to ensure sufficient H2O2 production without compromising the REEA signal (FIG. 3D). A ChOx concentration above 2 mg / mL resulted in a saturated response, with 10 mg / mL selected as the optimal concentration for subsequent experiments. Similarly, the concentration of CC in the injected solution was varied while maintaining constant levels of dried ChOx (10 mg / mL), AuNP- HRP-E2 (0.05 OD), and FP (5 mM) (FIG. 3E). Saturation was observed at CC concentrations above 1 mM. with 25 mM chosen as the optimal concentration. Finally. FP concentration was optimized under the conditions established in FIGS. 3D and 3E. FP concentrations above 0.5 mM led to signal saturation, and 5 mM was selected as the optimal concentration for further experiments (FIG. 3F).

[0092] In the next step, surfactants for the testing solution were screened in FIG. 4A by evaluating 24 different surfactants: TX-100, Tween 20, Surfactant 10G, Chemal LA-9, Aerosol® OT, Pluronic® L64. IGEPAL® CA610, Pluronic® F127, Pluronic® F68, BRIJ® 98, Triton® X-305, BRIJ® 35, Triton® X-45, Tween® 80, Tergitol®, Silwet® L7600, Synperonic® F108, Cremophor® EL, Tween® 60, Merpol® A, Benzalkonium, Tetronic® 90R4, 2,4,7,9-Tetramethyl-5-decindiol. The investigation focused on E2 capture efficiency, signal resolution, and proton generation efficiency, with each surfactant mixed into a 2% BSA in lx PBS solution. IGEPAL was selected for its ability to increase E2 capture efficiency by 21.8% and proton generation efficiency of REEA system by 31.06%, while maintaining good baseline control (CV: 3.04%). However, IGEPAL slightly reduced the signal resolution by 12.93%, where signal resolution is defined as the difference between the signal for a sample with no free E2 and the signal for the same solution spiked with 1 ng / mL of E2. To mitigate this, stabilizer components were screened as shown in FIG. 4B, including2.5% Polyvinylpyrrolidone, 1% Sucrose, 5% EtOH, 5% IP A, 1% Polyvinyl Alcohol, 1% Polyethylene glycol, 10% Acetonitrile, 1% Trehalose. This led to the selection of 2.5% PVP, which enhanced the signal resolution by 66.49%. The final optimized reagent comprised 2% BSA, 0.2% IGEPAL, and 2.5% PVP.

[0093] REEA cartridge for E2 Detection. The cartridge designed for E2 detection is divided into three primary zones: detection, bridge, and signal (FIG. 5A and FIG. 6). The process begins with the mixing of a plasma sample with AuNP-HRP-E2 (0. 1 OD), E2 antibody. CC (25 mM), and FP (5 mM) (Step 1). This mixture is incubated for 15 minutes to allow the competitive reaction to occur within the testing solution (Step 2). Due to its smaller size, free E2 has a higher binding affinity for the antibodies compared to the larger AuNP- HRP-E2 conjugates. After injection of the incubated sample into the main chamber, the sample flows through the detection zone — functionalized with secondary antibodies — where E2 antibodies bound to either AuNP-HRP-E2 or free E2 are captured. Within the detection zone of the cartridge strip, E2 antibodies — bound either to AuNP-HRP-E2 or free E2 — are captured by secondary' antibodies immobilized in this zone (Step 3). The unbound AuNP- HRP-E2 conjugates, together with CC and FP, then migrate across the bridge zone (Step 4) to the signal zone (Step 5), where choline oxidase (ChOx) is immobilized. In the signal zone, the first enzymatic reaction occurs between CC and ChOx, producing H2O2. This H2O2 initiates a second enzymatic reaction between HRP on the AuNP conjugate and FP, generating protons. These protons induce surface potential changes on the ITO electrode, which is remotely connected to the gate of the FET. The FET measures the surface potential of the ITO. providing the final detection signal.

[0094] Incubation is a critical step in enhancing signal resolution. The final detection signal is based on the filtration of HRP -AuNP, specifically detecting unbound HRP-AuNP in the detection zone. With incubation, more HRP-AuNP binds to E2 antibodies in the testing solution during Step 2 of FIG. 5 A. Consequently, more HRP-AuNP is retained in the detection zone, and less HRP-AuNP reaches the signal zone, leading to a lower signal. Without incubation, there is insufficient time for the formation of E2 antibody -HRP-AuNP complexes in the testing solution. As a result, HRP-AuNP flows nonspecifically into the signal zone, producing a higher signal. Due to this effect, there was an insignificant signal difference between samples containing 184 pg / mL and 576 pg / mL of E2. The signal resolution, determined by the difference in REEA signal between plasma samples containing184 pg / mL and 576 pg / mL of E2, saturated after 15 minutes of incubation. This was optimized as the ideal incubation time for the procedure.

[0095] Another critical parameter in the REEA system is the concentration of AuNP- HRP-E2 conjugates. When the concentration is too high, the binding sites of E2 antibodies during incubation become fully occupied, regardless of the free E2 concentration in the testing solution. To determine the optimal concentration of AuNP-HRP-E2 conjugates, the same paper fluidics setup was used with two testing solutions: one containing 5 ng / mL of free E2 and the other without free E2. The SNR was calculated by dividing the signal from the 5 ng / mL E2 sample by the signal from the sample with no E2, as a function of the AuNP-HRP- E2 conjugate concentration. An SNR of 1 indicates no signal differentiation between the two scenarios. Reducing the AuNP-HRP-E2 concentration corresponding to 0. 1 OD525 significantly increased the SNR to 5, which was identified as the optimal concentration for the competitive immunoassay (FIG. 5B).

[0096] Measurement time was optimized to balance signal resolution with detection speed, constrained by the enzymatic reaction duration. The cunent cartridge design, comprising detection, bridge, and signal generation zones, required 600 seconds to achieve precise signals at concentrations below 5 pg / mL. Notably, the maximum REEA signal was reached within 150-200 seconds, with approximately half of the total measurement time attributed to fluid flow through the detection and signal zones.

[0097] Under the optimized testing conditions, the E2-spiked sample was evaluated across a concentration range of 328 fg / mL to 1 ng / mL to determine the LOD of the system (FIG. 5C). The LOD was calculated to be approximately 146 fg / mL using the 3-sigma rule, with the CV for each E2 concentration being less than 9.2%. Additionally, clinical plasma samples with E2 concentrations ranging from 19 pg / mL to 4,551 pg / mL were tested (FIG. 5D), showing an r2of 0.963 and a standard error of the estimate (Sy / x) of 0. 15. as determined by logarithmic regression analysis when compared to results from FDA-cleared clinical immunoassay (FIG. 5D).

[0098] DISCUSSION

[0099] This Example presents a diagnostic platform that integrates REEA with a low-cost paper-based fluidic system ($0.55 per test) and a handheld FET reader. This system enables the quantification of protons for biomarker detection and consistently covers a broad analytical range of E2 concentrations from 19 to 4,551 pg / mL, achieving an r2of 0.963 withclinical plasma samples within 10 minutes. The platform also demonstrated a LOD of 146 fg / mL with a CV below 9.2% in E2-spiked buffer, effectively addressing the analytical sensitivity limitations typically associated with conventional at-home diagnostic platforms. Conversely, paper fluidics addresses the complexity of FET biosensors, eliminating the need for surface immobilization of biomolecules such as enzy mes, DNA, or antibodies. This paper fluidics system also enables low-cost production ($0.55 per test) and optimized LFA components have demonstrated a shelf life of up to two years.

[0100] A key advantage of the REEA platform is its ability to process clinical samples directly, bypassing the need of traditional FET biosensors for capture-release methods that typically require complex washing steps and buffer dilution to address Debye length challenges and matrix effects. This simplification allows for the use of paper-based fluidics, reducing system complexity.

[0101] The current detection time of 10 minutes, required for high signal resolution, can be reduced, as the maximum REEA signal is generated in approximately 200 seconds. The lateral paper strip design accounts for approximately 50% of the measurement time, primarily due to fluid passage. A vertical paper fluidics structure may be used to reduce wetting time while maintaining the capture efficiency.

[0102] Though primarily optimized for E2 detection, the REEA platform may be extended to a broad range of biomarkers, enhancing its versatility in personalized medicine and diagnostics. In line with the global shift towards home-based healthcare, the REEA platform addresses the critical need for accurate, cost-effective, and rapid diagnostics beyond traditional clinical settings. This development represents a significant step towards improving healthcare accessibility', supporting efforts to meet the growing demands on healthcare systems worldwide.

[0103] The word "illustrative" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as "illustrative" is not necessarily to be construed as preferred or advantageous over other aspects or designs. Further, for the purposes of this disclosure and unless otherwise specified, "a" or "an" means "one or more.”

[0104] The foregoing description of illustrative embodiments of the disclosure has been presented for purposes of illustration and of description. It is not intended to be exhaustive or to limit the disclosure to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the disclosure.The embodiments were chosen and described in order to explain the principles of the disclosure and as practical applications of the disclosure to enable one skilled in the art to utilize the disclosure in various embodiments and with various modifications as suited to the particular use contemplated. It is intended that the scope of the disclosure be defined by the claims appended hereto and their equivalents.

[0105] If not already included, all numeric values of parameters in the present disclosure are proceeded by the term “about’' which means approximately. This encompasses those variations inherent to the measurement of the relevant parameter as understood by those of ordinary skill in the art. This also encompasses the exact value of the disclosed numeric value and values that round to the disclosed numeric value.

[0106] Unless otherwise indicated, and in recognition of the inherent nature of the techniques described herein, throughout the present disclosure, terms and phrases such as “absence,” “free,” “does not comprise,” etc. encompass, but do not require a perfect absence of the referenced entity.

[0107] Unless otherwise indicated, the term “type” as used herein refers to chemical formula such that a single type means the same chemical formula and different t pe means different chemical formula. Similarly, use of “more” as in “one or more” refers to use of different types of the relevant entity.

[0108] Throughout the present disclosure, terms such as “comprising” and the like may be replaced with terms such as “consisting” and the like.

Claims

WHAT IS CLAIMED IS:1 . A system for electrically detecting a biomarker, the system comprising: a) a sample unit comprising: a sample chamber configured to contain a fluid sample; a reference electrode in fluid communication with the sample chamber; a detection zone in fluid communication with the sample chamber and configured to capture a biomarker; a signal zone in fluid communication with the detection zone; and a working electrode configured such that its surface potential changes in the presence of charged species generated by a reaction occurring in the signal zone; and b) a measuring unit comprising a field-effect transistor in electrical communication with the reference electrode and the working electrode, the measuring unit configured to measure a change in surface potential of the working electrode due to generated charged species.

2. The system of claim 1, wherein the charged species comprise protons and the reaction is between an enzy me and a radical substrate.

3. The system of claim 1, wherein the detection zone comprises immobilized secondary antibodies for capturing a primary antibody of the biomarker.

4. The system of claim 3, wherein the biomarker comprises a hormone, a protein, a chemical, a biological agent, a virus, or bacteria.

5. The system of claim 3, wherein the biomarker comprises a fertility' hormone.

6. The system of claim 2, wherein the signal zone is configured to induce the reaction between the enzyme and the radical substrate.

7. The system of claim 6, wherein the signal zone comprises immobilized enzymes that produce a peroxide.

8. The system of claim 7, wherein the immobilized enzymes comprise choline oxidase, glucose oxidase, or combinations thereof.

9. The system of claim 1, wherein the signal zone is provided by a porous substrate that is in direct contact with the working electrode and the signal zone and the working electrode are in fluid communication.

10. The system of claim 1, wherein the detection zone and the signal zone are provided by a porous substrate through which the fluid sample flows.

11. The system of claim 10, wherein the porous substrate comprises nitrocellulose, cotton, cellulose, glass fibers, polymer fibers, and combinations thereof.

12. The system of claim 1, further comprising a bridge zone in between the detection zone and the signal zone and in fluid communication with both.

13. The system of claim 1, wherein the working electrode comprises indium tin oxide (ITO), tin oxide, zinc oxide, a metal oxide semiconductor, graphene, reduced graphene oxide, a conductive polymer, or a conductive nanostructured material.

14. The system of claim 1, wherein the field-effect transistor is not in fluid communication with the detection zone and is not in fluid communication with the signal zone.

15. The system of claim 1, wherein the working electrode is in electrical communication with the gate of the field-effect transistor and the reference electrode is configured to apply a gate voltage to the field-effect transistor.

16. The system of claim 1 , further comprising a microcontroller unit in electrical communication with the field-effect transistor and configured to apply a drain voltage to the field-effect transistor and further configured to apply a gate voltage to the field-effect transistor via the reference electrode.

17. The system of claim 1, further comprising an operational amplifier assembly in electrical communication with the field-effect transistor and configured to measure an output voltage corresponding to the change in surface potential of the working electrode.

18. The system of claim 1, further comprising a display configured to display an output voltage corresponding to the change in surface potential of the working electrode or aconcentration value corresponding to a concentration of the biomarker in a test specimen or both, wherein the concentration value is determined from the output voltage.

19. The system of claim 1, wherein the sample unit and the measuring unit and configured to detach from one another; one or both of the sample unit and the measuring unit are configured to be held by a hand of a user; or both.

20. A method for electrically detecting a biomarker, the method comprising: a) flowing the fluid sample through the detection zone of the system of claim 1, the fluid sample comprising an enzy me, a radical substrate, a primary antibody for the biomarker, and a test specimen, wherein at least some biomarkers in the fluid sample are captured in the detection zone; b) flowing uncaptured components of the fluid sample to the signal zone, wherein reactions between uncaptured enzymes and uncaptured radical substrates generate protons; and c) measuring the change in surface potential of the working electrode due to generated protons using the field-effect transistor of the measuring unit.

21. The method of claim 20, wherein the enzyme is a component of a biomarkerenzyme conjugate comprising the biomarker and the enzyme.

22. The method of claim 21, wherein the biomarker-enzyme conjugate further comprises a nanoparticle having a surface on which multiple biomarker-enzyme complexes are bound.

23. The method of claim 20, further comprising forming the fluid sample by combining the enzyme, the radical substrate, the primary antibody for the biomarker, and the test specimen and incubating for an incubation time.

24. The method of claim 20, wherein the enzyme catalyzes oxidation of the radical substrate using a peroxide.

25. The method of claim 20, wherein the radical substrate is a phenolic compound.

26. The method of claim 25, wherein the phenolic compound is selected from fluorophenol, 4-iodophenoL 4-chlorophenol, 4-bromophenoL and combinations thereof.

27. The method of claim 20, wherein the fluid sample further comprises a reagent used by an enzyme to produce a peroxide.

28. The method of claim 27, wherein the reagent is choline chloride or glucose.

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