Cofactor-integrated biosensor

Abstract

The present embodiments relate to a cofactor-integrated biosensor architecture based on a single-wall-carbon-nanotubes framework, which simultaneously adsorbs cofactors, self-mediates cofactor redox reactions, and enhances reaction rates at the limit of enzyme activity. This framework supports detection / interference-elimination enzymes and encapsulation layers, achieving versatile biosensing with over 100-fold improved signal-to-noise ratio and days-long stability.

Description

COFACTOR-INTEGRATED BIOSENSOR CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 627,643 filed January 31, 2024, the contents of which are incorporated herein by reference in their entirety. STATEMENT OF GOVERNMENT SPONSORED RESEARCH

[0002] This invention was made with government support under DK128711 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The present embodiments relate generally to health and more particularly to a cofactor-integrated biosensor for in-vivo biomonitoring of a variety of biomarkers. BACKGROUND

[0004] Oxidoreductase enzymes are naturally well-suited for robust in-vivo biomonitoring. However, over 85% of the oxidoreductase library, mainly cofactor-based enzymes, remains unutilized due to challenges such as cofactor molecule incorporation and accuracy. It is against this technological backdrop that a technological solution to these and other problems rooted in this technology was sought. SUMMARY

[0005] The present embodiments relate to a cofactor-integrated biosensor. According to some aspects, embodiments allow for unlocking the oxidoreductase library for the purpose of in- vivo biomonitoring of a wide variety of biomarkers. 1 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151BRIEF DESCRIPTION OF THE DRAWINGS

[0006] These and other aspects and features of the present embodiments will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures, wherein:

[0007] Figs.1A to 1C illustrate example aspects of cofactor-integrated biosensing according to embodiments.

[0008] Figs.2A to 2J illustrate example characterizations of CIB performance according to embodiments.

[0009] Figs.3A to 3M illustrate example electrochemical characterizations of enzymatic CIBs according to embodiments.

[0010] Figs.4A, 4B(i) and 4B(ii) and 4C to 4J illustrate example aspects of in-vivo applications of CIBs for a mouse model and human subjects.

[0011] Fig.5 illustrates example aspects of rotating disk electrode measurement of NADH oxidation according to embodiments. Linear scanning voltammetry of the SWCNT-based NADH sensor at various rotational speeds.

[0012] Figs.6A and 6B illustrate example aspects of specific capacitance of SWCNT- based NADH sensor according to embodiments: (A) Cyclic voltammetry of the SWCNT-based NADH sensor at varying scanning rates. (B) Corresponding plot of current vs. scan rate at 0.1 V (vs. RHE).

[0013] Fig.7 illustrates example aspects of BHB response simulation according to embodiments. Simulated BHB calibration curve of the enzymatic electrochemical reaction hybrid model closely matches the experimental calibration curve in Fig.3I.

[0014] Figs.8A to 8G illustrate example aspects of SNR comparison for various analytes according to embodiments. (A-F) SNR comparison among GC, PD / CP, and CIB for metformin, acetaminophen, caffeine, uric acid, histidine, and tryptophan. (G) SNR comparison among GC, PD / CP, bare CIB, and AAOx-coupled-CIB for AA.

[0015] Figs.9A and 9B illustrate an example reversibility test according to embodiments. Real-time amperometric measurement with various concentrations of the analyte in 1× PBS suggested that the high level of reversibility of (A) glucose-CIB and (B) glutamate-CIB. 2 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151

[0016] Figs.10A to 10C illustrate an example real-time amperometric measurement with fouling agent according to embodiments.1000-minute real-time amperometric measurements performed with (A) BHB-CIB, (B) glucose-CIB, and (C) glutamate-CIB in 1× PBS buffer with 20 mg / mL BSA. The analyte concentrations are annotated for the corresponding time windows.

[0017] Figs.11A and 11B illustrate an example selectivity test with various electroactive species according to embodiments: (A) Real-time amperometric selectivity study with a representative glucose-CIB. (B) Real-time amperometric selectivity study with a representative glutamate-CIB. The introduction timepoints for the interferents and target analytes are indicated by the arrows.

[0018] Figs.12A and 12B illustrate example aspects of an ex-vivo biocompatibility test according to embodiments. The results of cellular viability test in terms of (A) fluorescence intensity (B) and fraction of viable cells.4 CIBs were immersed into 2 mL (2×) or 4 mL (×) of the medium. The cell toxicity was evaluated based on the human foreskin fibroblasts (HFF) system. Error bars indicate standard deviations. **** P < 0.0001.

[0019] Figs.13A to 13F illustrates examples aspects of in-vivo multiplexed CIBs monitoring according to embodiments. FIGs.13A, 13C and 13E illustrate three experiments with continuous monitoring during dual injection of BHB. Figs.13B, 13D and 13F Three experiments with continuous monitoring during one injection of glucose and one injection of BHB. Red arrows indicate time of collection of blood.

[0020] Fig.14 illustrates example aspects of SNR comparison among GC, PD / CP, bare CIB, and AAOx-coupled-CIB for AA in saliva according to embodiments. BHB-DH layer was drop-cast on to GC and PD / CP electrode to fabricate a BHB sensor. GC and PD / CP were test in saliva supplemented with 1 mM NAD+ to ensure sufficient NAD+ supply.

[0021] Figs.15A to 15C illustrate example aspects of BHB determination in sweat using wireless sensing system according to embodiments. (A) Photograph of the custom-developed wireless readout board next to a U.S. quarter. The components are (1) microcontroller unit, MCU, (2) potentiostat chip, and (3) Bluetooth chip. (B) BHB-CIB calibration response measured by the custom-developed readout board in 1× PBS buffer. (C) The determination of BHB concentration in sweat by CIB-based wireless sensing system vs. the corresponding standard assay-quantified BHB concentrations. 3 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151DETAILED DESCRIPTION

[0022] The present embodiments will now be described in detail with reference to the drawings, which are provided as illustrative examples of the embodiments so as to enable those skilled in the art to practice the embodiments and alternatives apparent to those skilled in the art. Notably, the figures and examples below are not meant to limit the scope of the present embodiments to a single embodiment, but other embodiments are possible by way of interchange of some or all of the described or illustrated elements. Moreover, where certain elements of the present embodiments can be partially or fully implemented using known components, only those portions of such known components that are necessary for an understanding of the present embodiments will be described, and detailed descriptions of other portions of such known components will be omitted so as not to obscure the present embodiments. Embodiments described as being implemented in software should not be limited thereto, but can include embodiments implemented in hardware, or combinations of software and hardware, and vice- versa, as will be apparent to those skilled in the art, unless otherwise specified herein. In the present specification, an embodiment showing a singular component should not be considered limiting; rather, the present disclosure is intended to encompass other embodiments including a plurality of the same component, and vice-versa, unless explicitly stated otherwise herein. Moreover, applicants do not intend for any term in the specification or claims to be ascribed an uncommon or special meaning unless explicitly set forth as such. Further, the present embodiments encompass present and future known equivalents to the known components referred to herein by way of illustration.

[0023] Real-time, continuous in-vivo biosensing is desirable for unraveling dynamic biological processes, such as metabolism, and enabling timely, effective intervention through health monitoring (W. Gao, S. Emaminejad, H. Y. Y. Nyein, S. Challa, K. Chen, A. Peck, H. M. Fahad, H. Ota, H. Shiraki, D. Kiriya, D.-H. Lien, G. A. Brooks, R. W. Davis, A. Javey, Fully integrated wearable sensor arrays for multiplexed in situ perspiration analysis. Nature 529, 509– 514 (2016); S. Wang, Y. Liu, A. Zhu, Y. Tian, In vivo electrochemical biosensors: Recent advances in molecular design, electrode materials, and electrochemical devices. Anal. Chem.95, 388–406 (2023)). To this end, harnessing the potential of oxidoreductase enzymes is a 4 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151promising strategy. These enzymes offer remarkable versatility for biosensing as they naturally catalyze a wide array of reactions in living organisms, including energy production, biosynthesis of essential molecules, and maintaining redox homeostasis (S. W. May, S. R. Padgette, Oxidoreductase enzymes in biotechnology: Current status and future potential. Bio / Technology 1, 677–686 (1983); F. Xu, Applications of oxidoreductases: Recent progress. Ind. Biotechnol.1, 38–50 (2005)). These reactions involve a diverse range of substrate molecules, notably metabolites and nutrients, which hold high physiological relevance as target biomarkers in biosensing applications. Oxidoreductase enzymes are also evolutionarily refined for delivering stable and specific catalytic reactions within complex biofluid environments, thus they are suitable for robust biosensing (H. H. Nguyen, S. H. Lee, U. J. Lee, C. D. Fermin, M. Kim, Immobilized enzymes in biosensor applications. Materials 12, 121 (2019)). Additionally, the redox reactions they facilitate align seamlessly with electrochemical signal transduction for real- time and continuous readouts.

[0024] Despite this substantial potential, the oxidoreductase library remains significantly underutilized for in-vivo biosensing. Since the introduction of enzymatic sensors in the 1960s, most demonstrations have been focused on oxidases (within the oxidoreductase library), which operate independently of cofactors to facilitate redox reactions (S. J. Updike, G. P. Hicks, The enzyme electrode. Nature 214, 986–988 (1967)). As shown by Fig.1A, this narrow focus excludes over 85% of the oxidoreductase library 102, comprising cofactor-dependent enzymes, the majority of which (e.g., dehydrogenases) rely on nicotinamide adenine dinucleotide (NAD+) as a cofactor for redox reactions (Fig.1(A)) (L. Sellés Vidal, C. L. Kelly, P. M. Mordaka, J. T. Heap, Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application. Biochim. Biophys. Acta BBA - Proteins Proteomics 1866, 327–347 (2018)). As further shown in Fig.1A, this underutilization drastically restricts the range of measurable biomarkers 104, considering that NAD-based enzymatic reactions can exclusively catalyze over 800 substrates (biosensing targets), which is an order of magnitude greater than what oxidase systems offer data in Fig.5). To summarize, Fig.1A illustrates classification of the oxidoreductase library 102 on the basis of cofactor dependency for catalytic reactions, their corresponding electroenzymatic sensing mechanisms, and projected measurable biomarkers 104. As further illustrated in Fig. 5 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-11511A, unlocking oxidoreductase library for in-vivo biomonitoring of such biomarkers enables clinical applications such as personalized metabolomics applications 106.

[0025] Cofactor-based biosensing, however, is more complex in implementation than oxidase-based biosensing. This complexity extends to both aspects of facilitating catalytic reactions (e.g. generating a signal) and countering interference from electroactive species (e.g. detecting the signal in presence of noise). Oxidase-based sensors rely solely on the abundant oxygen present in biofluids to drive reactions in the presence of target species (X. Cheng, B. Wang, Y. Zhao, H. Hojaiji, S. Lin, R. Shih, H. Lin, S. Tamayosa, B. Ham, P. Stout, K. Salahi, Z. Wang, C. Zhao, J. Tan, S. Emaminejad, A mediator-free electroenzymatic sensing methodology to mitigate ionic and electroactive interferents’ effects for reliable wearable metabolite and nutrient monitoring. Adv. Funct. Mater.30, 1908507 (2020)). Furthermore, their reaction end- product, hydrogen peroxide, is relatively small, allowing the straightforward application of size- based exclusion techniques (e.g., permselective membrane coating) for interference mitigation (Id.). In contrast, cofactor-based sensors do not benefit from easy access to cofactor molecules, as these molecules are either absent or present in very low concentrations in biofluids (due to their primary natural role being confined to intracellular reactions) (A. Nikiforov, V. Kulikova, M. Ziegler, The human NAD metabolome: Functions, metabolism and compartmentalization. Crit. Rev. Biochem. Mol. Biol.50, 284–297 (2015)). Additionally, the cofactor-based reaction end-products (e.g., reduced NAD+, NADH) are relatively large and require high overpotentials to drive the underlying oxidation reactions (W. J. Blaedel, R. A. Jenkins, Electrochemical oxidation of reduced nicotinamide adenine dinucleotide. Anal. Chem.47, 1337–1343 (1975); J. Moiroux, P. J. Elving, Mechanistic aspects of the electrochemical oxidation of dihydronicotinamide adenine dinucleotide (NADH). J. Am. Chem. Soc.102, 6533–6538 (1980); C. O. Schmakel, K. S. V. Santhanam, P. J. Elving, Nicotinamide adenine dinucleotide (NAD+) and related compounds. Electrochemical redox pattern and allied chemical behavior. J. Am. Chem. Soc.97, 5083–5092 (1975)). The former precludes the use of size-based exclusion techniques for selectivity enhancement, and the latter substantially intensifies electroactive- induced side reactions and concurrently contributes to electrode fouling through irreversible NAD+ decomposition (Id.). 6 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151

[0026] Remarkable progress has been made in the development of cofactor-based sensors, yet they fall short of meeting the stringent demands of in-vivo biosensing. As illustrated in Fig.1B, most demonstrations such as that illustrated by 108 rely on the external introduction of cofactors through biofluid spiking, which is incompatible with in-vivo sensing (R. Del Caño, T. Saha, C. Moonla, E. De la Paz, J. Wang, Ketone bodies detection: Wearable and mobile sensors for personalized medicine and nutrition. TrAC Trends Anal. Chem.159, 116938 (2023); C.-C. Wang, J. W. Hennek, A. Ainla, A. A. Kumar, W.-J. Lan, J. Im, B. S. Smith, M. Zhao, G. M. Whitesides, A paper-based “pop-up” electrochemical device for analysis of beta- hydroxybutyrate. Anal. Chem.88, 6326–6333 (2016)). Other approaches involve incorporating cofactors near the reaction site by embedding both cofactor molecules and enzymes within a polymeric membrane atop a carbon electrode (e.g., glassy carbon, GC, or carbon paste, CP) (J.- M. Moon, R. Del Caño, C. Moonla, K. Sakdaphetsiri, T. Saha, L. Francine Mendes, L. Yin, A.- Y. Chang, S. Seker, J. Wang, Self-testing of ketone bodies, along with glucose, using touch- based sweat analysis. ACS Sens.7, 3973–3981 (2022); H. Teymourian, C. Moonla, F. Tehrani, E. Vargas, R. Aghavali, A. Barfidokht, T. Tangkuaram, P. P. Mercier, E. Dassau, J. Wang, Microneedle-based detection of ketone bodies along with glucose and lactate: Toward real-time continuous interstitial fluid monitoring of diabetic ketosis and ketoacidosis. Anal. Chem.92, 2291–2300 (2020)). However, as illustrated by 110 in Fig.1B, such implementations fundamentally yield suboptimal reaction rates due to the limited catalytic efficiency / capacity of the electrodes and relatively large cofactor diffusion distances (micrometer-scale) that constrain mass transport.

[0027] Furthermore, as illustrated by 112 in Fig.1B, the reported cofactor-based sensors suffer from electroactive interference, even those employing mediators (e.g., 1,10- phenanthroline-5,6,-dione, PD, and its complexes) for lowering the cofactor oxidation potential (L. Gorton, Chemically modified electrodes for the electrocatalytic oxidation of nicotinamide coenzymes. J. Chem. Soc. Faraday Trans.1 Phys. Chem. Condens. Phases 82, 1245–1258 (1986); B. Prieto-Simón, E. Fàbregas, Comparative study of electron mediators used in the electrochemical oxidation of NADH. Biosens. Bioelectron.19, 1131–1138 (2004); L. Gorton, E.Domın ́guez, Electrocatalytic oxidation of NAD(P)H at mediator-modified electrodes. Rev. Mol.Biotechnol.82, 371–392 (2002); C. A. Goss, H. D. Abruna, Spectral, electrochemical and 7 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151electrocatalytic properties of 1,10-phenanthroline-5,6-dione complexes of transition metals. Inorg. Chem.24, 4263–4267 (1985)). For example, they are susceptible to interference from ascorbic acid (AA), which naturally fluctuates based on the subjects' nutritional intake (M. Wu, X. Mao, X. Li, X. Yang, L. Zhu, 1,10-phenanthroline-5,6-dione adsorbed on carbon nanotubes: The electrochemistry and catalytic oxidation of ascorbic acid. J. Electroanal. Chem.682, 1–6 (2012); R. Singh, A. A. Mahdi, R. K. Singh, C. Lee Gierke, G. Cornelissen, Effect of gender, age, diet and smoking status on the circadian rhythm of ascorbic acid (vitamin C) of healthy Indians. J. Appl. Biomed.16, 180–185 (2018); M. Levine, C. Conry-Cantilena, Y. Wang, R. W. Welch, P. W. Washko, K. R. Dhariwal, J. B. Park, A. Lazarev, J. F. Graumlich, J. King, L. R. Cantilena, Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc. Natl. Acad. Sci.93, 3704–3709 (1996)).

[0028] This interference results in significant inaccuracies, which is particularly troubling as AA is a key component of dietary interventions for subjects with metabolic disorders who could primarily benefit from cofactor-enabled biomonitoring modalities (M. G. Traber, G. R. Buettner, R. S. Bruno, The relationship between vitamin C status, the gut-liver axis, and metabolic syndrome. Redox Biol.21, 101091 (2019); E. Sawicka-Glazer, S. J. Czuczwar, Vitamin C: A new auxiliary treatment of epilepsy? Pharmacol. Rep.66, 529–533 (2014)). These sensors are also susceptible to the leakage of their constituent components, such as mediators and cofactor molecules, leading to unstable and irreversible responses and restricting measurements to a single time point or short time window (C. Moonla, R. Del Caño, K. Sakdaphetsiri, T. Saha, E. De la Paz, A. Düsterloh, J. Wang, Disposable screen-printed electrochemical sensing strips for rapid decentralized measurements of salivary ketone bodies: Towards therapeutic and wellness applications. Biosens. Bioelectron.220, 114891 (2023); X. Zhang, Y. Xia, Y. Liu, S. M. Mugo, Q. Zhang, Integrated wearable sensors for sensing physiological pressure signals and β- hydroxybutyrate in physiological fluids. Anal. Chem.94, 993–1002 (2022)).

[0029] The present embodiments relate to a cofactor-integrated biosensor (CIB) architecture that leverages various distinct features of single-wall carbon nanotubes (SWCNT) as the electrode framework to simultaneously render multiple functions essential for high- performance and versatile enzymatic sensing. Fig.1B further illustrates a comparison of the 8 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151conventional approach versus CIB-based dehydrogenase enzymatic sensing of the present embodiments.

[0030] For example, as shown by 114 in Fig.1B, through direct adsorption of cofactor molecules and utilizing SWCNT's high aspect ratio and superior electrocatalytic capabilities, this electrode framework reaches the theoretical limit of the overall reaction rate set by the enzyme's redox rate as shown by 116. It also possesses self-mediating capabilities for driving cofactor oxidation at 0 V (vs. Ag / AgCl, silver / silver chloride), dramatically minimizing electrode fouling and electroactive interference as shown by 118 in Fig.1B. This in turn eliminates the need for mediators and their associated challenges.

[0031] Furthermore, this electrode framework is versatile, permitting the incorporation of diverse functional layers. Fig.1C is an exploded view of an example CIB architecture and an image of a representative CIB array 122 according to embodiments. As shown in this example, the CIB 120 may be included as one of a plurality of sensors in the array 122. As further shown in Fig.1C, example CIB includes a plurality a plurality of layers 120 provided on an electrode base 134 (e.g. gold). The layers can include SWCNT electrodes 124 and 128 and distinct enzyme layers (e.g.126) for detection and supplemental interference elimination, as well as an encapsulation layer (e.g.132) for stabilization and anti-fouling. Importantly, the architecture accommodates the layering of detection and interference-elimination enzymes (e.g.130) to specifically catalyze target-induced reactions and consume the dominant interference sources (here, AA)—with no reaction crosstalk. Additionally, the electrodes’ (e.g.124 and 128) high specific area increases loading of cofactor molecules and detection / interference-elimination enzymes for enhanced signal-to-noise ratio (SNR) measurements.

[0032] Upon adapting this architecture for NAD-based sensing, developed was a library of CIBs for broad enzymatic sensing (for example, using 9 dehydrogenases targeting different biomarkers). These sensors demonstrated exceptional signal-to-noise ratios (> 100-fold higher than alternatives), and maintained high levels of reversibility (Δ < 7.5% across varying target concentrations) and stability (Δ < 7% over 3 days of continuous operation).

[0033] The CIBs are expanded into an array format (e.g. as shown by 122 in Fig.1C, including a plurality of CIB sensors and a reference electrode 138) for multiplexing and further employed in in-vivo biomonitoring. Demonstrated is CIB’s utility for enabling two distinct 9 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151physiological applications. First, the CIB platform (e.g.120, 122) was applied to study dynamic metabolic interactions in small animal models—addressing the limitations of poor temporal resolutions caused by sample collection challenges by enhancing the biomarker data sampling rate by two to three orders of magnitude. The CIB platform was also utilized for non-invasive health monitoring in both healthy subjects and patients with metabolic disorders through sweat and saliva analysis. The high signal-to-noise ratio of CIB uniquely overcame the biosensing reliability issues caused by secretion-induced metabolite dilution (~10-fold) and interference.

[0034] By unlocking the oxidoreductase library for in-vivo biomonitoring of a wide spectrum of analytes, including vital metabolites and nutrients, the CIB platform creates new research and healthcare strategies for personalized metabolomics and precision nutrition.

[0035] Results

[0036] NAD+ can be integrated into the SWCNT substrate through direct absorption and leveraging π-π stacking interactions. This integration was visualized using Scanning Transmission Electron Microscopy (S / TEM) in conjunction with energy dispersive X-ray spectroscopy (EDS). Fig.2A provide example S / TEM-EDS images of CIB, including a high- angle annular dark-field (HAADF) image 202, an EDS image 204 for P, phosphorus, the signature element for NAD+, and an EDS image 206 for C, carbon. More particularly 202 depicts the morphology of the NAD+-integrated SWCNT (serving as our CIB framework), while the corresponding EDS images 204 and 206 indicate a consistent and concentrated distribution of NAD+ across the SWCNT substrate. The successful immobilization of NAD+ was further verified via cyclic voltammetry (CV)(R.-D. Nagarajan, P. Murugan, A.-K. Sundramoorthy, Selective electrochemical sensing of NADH and NAD+ using graphene / tungstate nanocomposite modified electrode. ChemistrySelect 5, 46 (2020)), revealing the distinctive redox signatures of NAD+ (Fig.2(B)). Fig.2B is a graph illustrating example aspects of CV characterization of CIB in PBS according to embodiments. As shown in Fig.2B, the presence of NAD+ is detected by CIB through a reduction 208 in current across a range of voltages.

[0037] Efficient oxidation of NADH, the byproduct of dehydrogenase-based reactions, is crucial for high-performance sensing. Fig.2C is an example schematic of NADH oxidation and decomposition reactions according to embodiments. As shown in Fig.2C, this reaction 10 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151theoretically involves a 2-electron transfer process 212, as represented by the electron transfer number, ne, of 2 (I. Katakis, E. Domínguez, Catalytic electrooxidation of NADH for dehydrogenase amperometric biosensors. Microchim. Acta 126, 11–32 (1997); P. J. Elving, C. O. Schmakel, K. S. V. Santhanam, P. Zuman, Nicotinamide-NAD sequence: Redox processes and related behavior: Behavior and properties of intermediate and final products. C R C Crit. Rev. Anal. Chem.6, 1–67 (1976)). Nonetheless, empirically, due to NADH’s inherent instability, which results in its irreversible decomposition 210 and electrode fouling (R. D. Braun, K. S. V. Santhanam, P. J. Elving, Electrochemical oxidation in aqueous and nonaqueous media of dihydropyridine nucleotides NMNH, NADH, and NADPH. J. Am. Chem. Soc.97, 2591–2598 (1975)), the effective electron transfer number is reduced. For conventional GC and PD / CP electrodes, ne is close to 1 (G. Hilt, T. Jarbawi, W. R. Heineman, E. Steckhan, An analytical study of the redox behavior of 1,10-phenanthroline-5,6-dione, its transition-metal complexes, and its N-monomethylated derivative with regard to their efficiency as mediators of NAD(P)+ regeneration. Chem. – Eur. J.3, 79–88 (1997); S. Immanuel, R. Sivasubramanian, Electrochemical studies of NADH oxidation on chemically reduced graphene oxide nanosheets modified glassy carbon electrode. Mater. Chem. Phys.249, 123015 (2020)).

[0038] The present CIB, based on acid-treated SWCNT, demonstrates significantly higher NADH oxidation efficiency with an ne as high as 1.90 ± 0.07 (obtained via rotating disk electrode, RDE, analysis, Fig.5), while also exhibiting high NADH sensitivity at 0 V (vs. Ag / AgCl) oxidation potential (i.e. self-mediating reactions). Fig.2D provides an example NADH calibration curve obtained from CIB (N = 3) with an inset 214 showing the 150 μM NADH oxidation signal stability comparison with other commonly used electrodes (e.g. GC and PD / CP). This enhanced performance can be attributed to the high specific area of the SWCNT substrate, the presence of quinone-based groups on the acid-treated SWCNT (L. Feng, H.-P. Li, K. Galatsis, H. G. Monbouquette, Effective NADH sensing by electrooxidation on carbon- nanotube-coated platinum electrodes. J. Electroanal. Chem.773, 7–12 (2016)), and the catalytic property of the SWCNT's edge plane (C. E. Banks, R. G. Compton, Exploring the electrocatalytic sites of carbon nanotubes for NADH detection: an edge plane pyrolytic graphite electrode study. Analyst 130, 1232–1239 (2005)). Fig.2E is a chart providing an example capacitance comparison among GC, PD / CP, and CIB (N = 3). Furthermore, the improved 11 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151oxidation efficiency of the CIB contributes to its superior anti-fouling performance. As illustrated by the results of our anti-fouling study (e.g.1-hour oxidation operation at 150 μM NADH, shown in inset 214), the degradation in the CIB's NADH response is less than one percent, which is over 10-fold smaller than that observed in other alternative electrodes (e.g. GC and PD / CP as shown in 214).

[0039] The CIB architecture of the present embodiments also enhances both the mass transport of the enzymatic reaction product and associated reaction kinetics, ultimately achieving optimal overall reaction rates. In CIB, cofactor molecules (NAD+) are directly immobilized onto the porous electrode substrate with a large specific area (e.g. A0, CIB ~ 108 / m). This design reduces the diffusion distance of the enzyme product (NADH) to the substrate electrode, down to a few nanometers. In contrast, conventional cofactor-based enzymatic sensors superficially immobilize cofactors atop the electrode substrate, within the enzyme layer of approximately a few micrometers thickness (X. Chen, N. Matsumoto, Y. Hu, G. S. Wilson, Electrochemically mediated electrodeposition / electropolymerization to yield a glucose microbiosensor with improved characteristics. Anal. Chem.74, 368–372 (2002)). Consequently, their diffusion length scales are on the order of micrometer-scale. Furthermore, the CIB drives NADH oxidation at a substantially higher intrinsic rate than conventional electrodes, as evidenced by its large exchange current density (i0,CIB ~ 17.4 ± 0.3 A / m2, Fig.5, as compared to i0,GC ~ 0.06 A / m2and i0,PD / CP ~ 0.35 A / m2)(F. Pariente, E. Lorenzo, H. D. Abruna, Electrocatalysis of NADH oxidation with electropolymerized films of 3,4-dihydroxybenzaldehyde. Anal. Chem.66, 4337– 4344 (1994)). This, together with CIB’s extremely large specific area as illustrated by Fig.2E, allows CIB to drive overall catalytic reactions (within a fixed footprint) at a dramatically higher capacity than alternative electrodes (see Figs.6A and 6B).

[0040] To study the CIB’s enhanced performance for enzymatic sensing, utilized was a finite element analysis-based simulation model. Within this model, key parameters such as cofactor arrangement, enzyme activity, and electrode properties / design (e.g., i0, A0), can be explored. First validated was the fidelity of the model by verifying its alignment with empirical measurements (Fig.7). Then, to study the effect of mass transport limitation, simulated was the transduced NADH oxidation current resulting from different NADH diffusion distances across three types of electrodes: SWCNT (CIB's framework), GC, and PD / CP (operated at their 12 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151intended oxidation voltages). Fig.2F provides example simulation results for 200 μM NADH oxidation currents with different diffusion distances for GC, PD / CP, and CIB. As shown in Fig. 2F, the results indicate that CIB transduces over 100-fold larger current. Next studied were the reaction kinetics of the electrodes in terms of their specific area and exchange current density. To conduct this study in isolation from mass transport limitations, the model was reconfigured to simulate NADH oxidation (generated by a model dehydrogenase reaction) directly taking place at the electrodes’ surfaces. The corresponding simulation results for a range of hypothetical exchange current densities and specific areas are shown in Fig.2G. More particularly, Fig.2G provides simulation results for 200 μM beta-hydroxybutyrate acid (BHB) signal currents with different exchange current densities under different electrode specific areas. They specifically indicate that not only does the CIB substantially outperform alternative GC and PD / CP electrodes, but it also facilitates catalytic reaction at a rate only limited by the enzyme activity (as evident from plateaued current response). Consistently observed was this performance achievement across a wide range of enzyme activities. More particularly, Fig.2H provides example simulation results for 200 μM BHB signal currents with different exchange current densities under different enzyme activities.

[0041] The CIB's self-mediating capability and its effective integration of an interference-elimination enzymatic layer allow for minimizing electroactive interference and achieving reactions with a high SNR, thereby addressing a fundamental challenge in enzymatic sensing.

[0042] To illustrate the self-meditation benefit, characterized was the CIB’s sensitivity to NADH and a panel of electroactive molecules commonly found in biofluids (S. Lin, B. Wang, W. Yu, K. Castillo, C. Hoffman, X. Cheng, Y. Zhao, Y. Gao, Z. Wang, H. Lin, H. Hojaiji, J. Tan, S. Emaminejad, Design framework and sensing system for noninvasive wearable electroactive drug monitoring. ACS Sens.5, 265–273 (2020); S. Lin, B. Wang, Y. Zhao, R. Shih, X. Cheng, W. Yu, H. Hojaiji, H. Lin, C. Hoffman, D. Ly, J. Tan, Y. Chen, D. Di Carlo, C. Milla, S. Emaminejad, Natural perspiration sampling and in situ electrochemical analysis with hydrogel micropatches for user-identifiable and wireless chemo / biosensing. ACS Sens. (2019); S. Lin, W. Yu, B. Wang, Y. Zhao, K. En, J. Zhu, X. Cheng, C. Zhou, H. Lin, Z. Wang, H. Hojaiji, C. Yeung, C. Milla, R. W. Davis, S. Emaminejad, Noninvasive wearable electroactive 13 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151pharmaceutical monitoring for personalized therapeutics. Proc. Natl. Acad. Sci.117, 19017– 19025 (2020)). For comparison, conducted was the same procedure using GC and PD / CP electrodes, and used was the ratio of the electrodes’ reaction sensitivity to NADH vs. interfering analytes as a measure of SNR. This definition of SNR offers comprehensive coverage across a spectrum of target and interference concentrations, ensuring applicability to diverse sensing scenarios. It is distinct from traditional single-point interference characterization methods, which often involve large concentration differences between the target and interfering molecules. Fig. 2I provides an example normalized SNR comparison among GC, PD / CP, and CIB against a panel of electroactive interferences in biofluids. Fig.2I demonstrates that for all interference cases, the CIB exhibited significantly higher SNR compared to the alternatives (Figs.8A to 8G).

[0043] The use of an interference-elimination enzyme layer is particularly applicable in mitigating AA interference, a predominant noise source that corrupts the responses of existing cofactor-based enzymatic sensors. The CIB’s exceptionally large specific area makes it suitable for immobilizing ascorbic acid oxidase (AAOx) with high loading to effectively counter this challenge. Followed was the aforementioned characterization procedure to study and benchmark the SNR performance of the AAOx-coupled CIB against AA interference. Fig.2J provides an example normalized SNR comparison among GC, PD / CP, bare CIB, and AAOx-coupled-CIB against AA. ****** P < 0.000001, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and “ns” denotes statistical non-significance. The error bars in Figs.2I and 2J indicate standard deviations (N = 3 for each electrode). Fig.2J shows that this strategy was extremely effective, as evidenced by the AAOx-coupled CIB’s 100-fold larger SNR compared to both the bare CIB and other electrodes (see also Figs.8A to 8G).

[0044] Transformed was the CIB into a versatile biosensing platform by incorporating detection-enzymatic layers. Nine dehydrogenases were separately introduced into the CIB design, each targeting a distinct analyte: glucose, L-glutamate, glucose 6-phosphate (G6P), ethanol, D-lactate, L-leucine, cholesterol, glycerol, and β-hydroxybutyrate (BHB). Concentration calibration plots for each enzymatic CIB were obtained through amperometric measurements conducted within the respective physiological concentration ranges of the analytes (as detailed in table 5). Figs.3A to 3I illustrate that all CIBs exhibited consistent, monotonic responses to analyte concentrations with reproducible sensitivities and minimal inter-device variations. More 14 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151particularly, Figs.3A to 3I provide CIBs calibration responses targeting glucose (Fig.3A), L- glutamate (Fig.3B), G6P (Fig.3C), ethanol (Fig.3D), D-lactate (Fig.3E), L-leucine (Fig.3F), cholesterol (Fig.3G), glycerol (Fig.3H), and BHB (Fig.3I). N = 3 for each tested CIB, and error bars indicate standard deviations.

[0045] The incorporation of an encapsulation layer such as 132 illustrated in Fig.1C (for example, polyvinyl chloride, PVC), within the CIB design, combined with the robust immobilization of cofactor molecules on the CIB's substrate, ensures the stability and reversibility of the enzymatic CIB’s response. To demonstrate the enzymatic CIB’s robustness, conducted was a prolonged characterization study, continuously recording the CIB’s response in a phosphate-buffered saline (PBS) buffer. Fig.3J illustrates a 3-day long real-time amperometric measurement performed with BHB-CIBs in PBS supplemented with a 2 mM BHB increase from baseline (0 mM). Normalized response = (I – IBaseline) / (IMax – IBaseline), error band indicates standard deviation (N = 3). As shown in Fig.3J, the CIB exhibited minimal response deviation, remaining within a few percentages, even after 3 days of continuous operation, indicating negligible cofactor leakage.

[0046] The reversibility of CIB was assessed by repeatedly immersing representative BHB, glucose, and glutamate-CIBs in solutions with increasing or decreasing target concentrations and continuously recording their responses at each concentration level. In all cases, the CIBs consistently adjusted to the expected response levels, with changes of less than 7.5% for each introduced concentration (Fig.3K and Figs.9A and 9B). Fig.3K illustrates real- time BHB amperometric responses with a representative BHB-CIB. Also assessed were these CIBs’ anti-fouling capability through continuous measurements in a protein-rich environment (e.g. PBS buffer with 20 mg / ml bovine serum albumin, BSA (S. Lin, X. Cheng, J. Zhu, B. Wang, D. Jelinek, Y. Zhao, T.-Y. Wu, A. Horrillo, J. Tan, J. Yeung, W. Yan, S. Forman, H. A. Coller, C. Milla, S. Emaminejad, Wearable microneedle-based electrochemical aptamer biosensing for precision dosing of drugs with narrow therapeutic windows. Sci. Adv.8, eabq4539 (2022))). During 1000-minute studies involving varying target concentrations, the enzymatic CIB responses’ declines were within 2% at each level (Figs.10A to 10C).

[0047] The enzymatic CIBs also possess exceptional selectivity toward the respective targets, owing to the specificity of the CIB electrode in catalyzing underlying NADH reactions 15 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151(governed by self-mediation and interference-elimination layer) and utilization of naturally selective enzymes for analyte recognition. To illustrate this point, recorded were the representative CIBs’ responses to a panel of progressively introduced interference molecules. Table 1 provide a listing of common interferences in biofluids (e.g., sweat and saliva). TABLE 1 Number 1 2 3 4 5 6 Interference Glucose Lactate KCI NaCl Uric Acid AA Conc. (uM) 50 5000 2400 10000 59 100

[0048] As listed in Table 1, the interference panel includes small molecules, ionic species, and electroactive species at their physiologically-relevant concentrations, with AA (dominant interference) tested at a high concentration (100 μM, compared to 50 μM, high end of salivary AA concentration) (M. Bariya, H. Y. Y. Nyein, A. Javey, Wearable sweat sensors. Nat. Electron.1, 160–171 (2018)). Fig.3M illustrates real-time amperometric selectivity study with a representative BHB-CIB sensor. As shown in Fig.3L and Figs.11A and 11B, the CIBs exhibited negligible response against the interference group. The results also illustrate there is no reaction crosstalk between the two enzymatic layers (i.e. interference-elimination and detection enzymes).

[0049] Fabricated was an array of CIBs (e.g. array such as 122 in Fig.1C) sharing a same reference electrode onto a soft substrate (e.g. styrene–ethylene–butylene–styrene block copolymer, SEBS) for multiplexed analyte monitoring. Fig.3M illustrates multiplexed real-time amperometric measurements in undiluted porcine serum from a panel of CIBs such as array 122 targeting BHB, glucose, L-glutamate, G6P, ethanol, D-lactate, and L-leucine. Dotted lines represent transient stabilization period post-analyte spiking. Tested was this CIB array’s response in a serum buffer by concurrently recording the amperometric measurements of all 7 channels and intermittently introducing individual analyte targets. As shown in Fig.3M, the sensors stably responded to their corresponding analytes with no detectable crosstalk.

[0050] Collectively, the ex-vivo characterization results support the high level of adaptability, sensitivity, selectivity, stability, and reversibility of the CIB platform for in-vivo 16 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151biomonitoring. Among many potential applications, this platform particularly enables dynamic molecular interaction studies in animal models and health monitoring in humans.

[0051] Regarding the first enabling application, it is worth noting that the majority of molecular interaction studies are conducted in small animals such as mice and rats (E. C. Bryda, The mighty mouse: The impact of rodents on advances in biomedical research. Mo. Med.110, 207–211 (2013)). These studies are confined by the sampling volume threshold of approximately 10% of the animal’s total circulating blood volume every two to four weeks (S. Parasuraman, R. Raveendran, R. Kesavan, Blood sample collection in small laboratory animals. J. Pharmacol. Pharmacother.1, 87–93 (2010)), limiting temporal resolutions to a few hours / days (even when utilizing low-volume bioanalytical methods such as liquid chromatography–mass spectrometry, LC–MS). Additionally, frequent sampling would confound physiological interpretations by triggering physiological responses such as stress, which can lead to metabolite fluctuations (e.g., blood glucose levels elevation) (M. R. Kennard, L. F. Daniels Gatward, A. G. Roberts, E. R. P. White, M. Nandi, A. J. F. King, The use of mice in diabetes research: The impact of experimental protocols. Diabet. Med.38, e14705 (2021)). The CIB platform can address these issues by generating in-vivo biomarker data at minute-level intervals, achieving data sampling rates two to three orders of magnitude higher than current methods relying on sample collection and ex-situ analysis.

[0052] Figs.4A to 4J illustrate example aspects of in-vivo applications of CIBs for a mouse model and human subjects. More particularly, Fig.4A provides schematic illustrations of metabolic interaction studies in a mouse model. As illustrated by 402 in Fig.4A, an in-vivo CIB array provides real-time continuous measurements (e.g. current response changes). Meanwhile, as illustrated by 404 in Fig.4A, collected mouse blood samples are provided for ex- situ assay analysis. The measurements are compared in connection with injection of target analytes via mouse tail-vein injection 406.

[0053] After validating the CIB’s biocompatibility through cellular viability studies (Figs.12A and 12B), monitored were blood BHB and glucose in mice using our multiplexed CIBs, which additionally included a negative control CIB (without detection enzyme). Figs. 4B(i) and 4B(ii) provides In-vivo real-time blood BHB and glucose monitoring in mice with multiplexed CIBs functionalized for BHB, glucose and negative control. Blood samples were 17 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151extracted at red-arrows-indicated time points for standard fluorometric assay quantifications. The shaded green and blue bands indicate the BHB and glucose tail-vein injection time windows respectively. Normalized current = (I – IBaseline) / IBaseline..As illustrated in Figs.4B(i) and 4B(ii), upon the administration of intravenous BHB or glucose, both BHB and glucose CIBs promptly captured the dynamic changes of the corresponding analytes, while the negative control maintained its baseline response. Figs.4C and 4D illustrate the in-vivo CIB-measured BHB (Fig. 4C) and glucose (Fig.4D) current response changes in mouse blood versus their corresponding ex-situ assay analysis of collected blood samples at the studies’ two endpoints. The observed analyte trends closely matched the ex-situ assay analysis of collected blood samples at the studies’ two endpoints (Figs.4C and 4D, R2BHB = 0.97 and R2glucose = 0.86). It's also worth noting that, consistent with prior studies, variable glucose trends were observed post BHB injections (Figs.13A to 13F) (S. L. Kesl, A. M. Poff, N. P. Ward, T. N. Fiorelli, C. Ari, A. J. Van Putten, J. W. Sherwood, P. Arnold, D. P. D’Agostino, Effects of exogenous ketone supplementation on blood ketone, glucose, triglyceride, and lipoprotein levels in Sprague– Dawley rats. Nutr. Metab.13, 9 (2016); C. Ari, C. Murdun, A. P. Koutnik, C. R. Goldhagen, C. Rogers, C. Park, S. Bharwani, D. M. Diamond, M. S. Kindy, D. P. D’Agostino, Z. Kovács, Exogenous ketones lower blood glucose level in rested and exercised rodent models. Nutrients 11, 2330 (2019); H. Y. Cha, S. J. Yang, The alterations of blood glucose and cognitive function by ketone injection in mice. FASEB J.34, 1–1 (2020)), requiring future focused investigations of metabolic interaction dynamics, which can be facilitated with the CIB platform.

[0054] For human health monitoring, utilized was the CIB platform to track metabolite profiles in non-invasively retrievable biofluids such as sweat and saliva. Non-invasive biomonitoring of metabolites in these biofluids offers a painless and wearable solution for wellness monitoring and disease management (S. Emaminejad, W. Gao, E. Wu, Z. A. Davies, H. Yin Yin Nyein, S. Challa, S. P. Ryan, H. M. Fahad, K. Chen, Z. Shahpar, S. Talebi, C. Milla, A. Javey, R. W. Davis, Autonomous sweat extraction and analysis applied to cystic fibrosis and glucose monitoring using a fully integrated wearable platform. Proc. Natl. Acad. Sci.114, 4625– 4630 (2017)), particularly for individuals with metabolic disorders such as diabetes or in need of modified diets like epilepsy. This capability aids them in tracking their daily nutritional intake and actively managing the risk of conditions such as diabetic ketoacidosis. However, this 18 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151biomonitoring modality is challenging due to the substantial dilution of metabolites in the secreted biofluids (typically entail over 10 times dilution upon partitioning from blood (P. Abikshyeet, V. Ramesh, N. Oza, Glucose estimation in the salivary secretion of diabetes mellitus patients. Diabetes Metab. Syndr. Obes.5, 149–154 (2012); L. B. Baker, A. S. Wolfe, Physiological mechanisms determining eccrine sweat composition. Eur. J. Appl. Physiol.120, 719–752 (2020))). Fig.4E provides schematic illustrations of ketosis metabolic pathways in the human body 412, and the diffusion of metabolites from blood to non-invasively retrievable biofluids (e.g., sweat and saliva) 414. The diluted metabolite concentrations fall below the sensors’ detection limits and / or make the sensors increasingly susceptible to interference fluctuations (especially from AA). However, the CIB platform is not prone to these issues, given its low limit of detection and ability to provide high SNR metabolite measurements (Fig.14).

[0055] To this end, first conducted were human subject studies to examine the correlation of metabolite levels in sweat and saliva with respect to their circulating levels. Here, experiments focused on ketone (specifically, BHB) due to its significance in precision nutrition applications such as personalized ketogenic diet planning and diabetes management (J. C. Newman, E. Verdin, β-Hydroxybutyrate: A signaling metabolite. Annu. Rev. Nutr.37, 51–76 (2017); J. Huang, A. M. Yeung, R. M. Bergenstal, K. Castorino, E. Cengiz, K. Dhatariya, I. Niu, J. L. Sherr, G. E. Umpierrez, D. C. Klonoff, Update on measuring ketones. J. Diabetes Sci. Technol., 19322968231152236 (2023); I. D’Andrea Meira, T. T. Romão, H. J. Pires do Prado, L. T. Krüger, M. E. P. Pires, P. O. da Conceição, Ketogenic diet and epilepsy: What we know so far. Front. Neurosci.13 (2019)). Sampled were saliva and sweat (following standard protocols) from two distinct cohorts: epileptic patients on a ketogenic diet and healthy subjects who had consumed a ketone supplement. The samples were subsequently analyzed using the BHB-CIB and commercialized fluorescent assays. The studies revealed strong correlations between BHB concentrations in saliva and blood and between sweat and blood in both cohorts, also validating our CIB’s high degree of accuracy in analyzing sweat and saliva BHB concentrations). Figs.4F and 4G provide saliva-blood (Fig.4F) and sweat-blood (Fig.4G) BHB concentration correlations for healthy subjects with a ketone supplement and epileptic patients under ketogenic diets. Figs. 4Hand 4I provide ex-situ CIB-measured BHB in human saliva (Fig.4H) and sweat (Fig.4I) samples versus their corresponding standard assay-quantified BHB concentrations. Also 19 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151validated was that the CIB readouts can be recorded by low power consumer electronics for wireless operation (Fig.15A).

[0056] Following the correlation studies, utilized was the CIB to track dynamic changes in metabolic states within the human body through salivary BHB analysis. Specifically investigated was its ability to detect metabolic state transition between ketosis and glycolysis. In ketosis, the body lacks the preferred energy source, glucose, therefore generates energy from stored fat, leading to elevated ketone levels (see Fig.4E) (Id.). In glycolysis, rising blood glucose stimulates the secretion of insulin to facilitate the intracellular glucose uptake, thereby strongly inhibiting ketosis and favoring glucose as the primary energy source (J. H. Ellenbroek, L. van Dijck, H. A. Töns, T. J. Rabelink, F. Carlotti, B. E. P. B. Ballieux, E. J. P. de Koning, Long-term ketogenic diet causes glucose intolerance and reduced β- and α-cell mass but no weight loss in mice. Am. J. Physiol.-Endocrinol. Metab.306, E552–E558 (2014)).

[0057] Fig.4J illustrates saliva BHB monitoring with BHB-CIB and blood glucose monitoring with commercialized glucose meter for a fasting healthy subject before and after consuming a high carbohydrate beverage. (P < 0.01 for data in Figs.4C, 4D and Figs.4F-4I) Fig.4J illustrates that the CIB captured an elevated salivary BHB concentration in a fasting healthy subject, indicating a metabolic state of ketosis. After the consumption of a carbohydrate- rich beverage, the BHB concentration dropped sharply, from approximately 25 μM to about 100 μM within 100 minutes (curve 422), suggesting a metabolic shift from ketosis to glycolysis. This transition was corroborated by the elevation of the glucose levels obtained by standard capillary blood analysis (curve 424).

[0058] Discussion

[0059] The CIB design of the present embodiments unlocks the rich library of oxidoreductases for in-vivo biomonitoring, making over 800 analytes accessible, including vital metabolites and nutrients. It enables the use of existing commercially-available enzymes for biosensing and promotes new directions for enzyme engineering and production for both optimal operation and new targets. Owing to its exceptionally high SNR, stability, reversibility, and capacity for multiplexing, CIB is adaptable to a variety of in-vivo biomonitoring modalities. 20 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151These render CIB a powerful platform for studying and monitoring metabolic interactions and health states, empowering personalized metabolomics and precision nutrition.

[0060] Example Materials and Methods

[0061] Example Materials

[0062] Single-walled carbon nanotubes (SWCNT), reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide (NAD+), metformin, uric acid (UA), caffeine, acetaminophen (APAP), L-histidine, L-tryptophan, L-ascorbic acid (AA), DL-β- hydroxybutyric acid sodium salt (BHB), D-(+)-glucose, L-glutamic acid, D-glucose 6-phosphate sodium salt, L-leucine, glycerol, sodium D-lactate, potassium chloride, sodium chloride, silver nitrate (AgNO3), bovine serum albumin (BSA), glutaraldehyde solution (25 wt%), polyvinyl chloride (PVC), tetrahydrofuran (THF), ethyl alcohol, and alcohol dehydrogenase were purchased from Sigma-Aldrich (MO, USA). D-3-hydroxybutyrate dehydrogenase, glucose dehydrogenase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, D-lactate dehydrogenase, cholesterol dehydrogenase, leucine dehydrogenase, glycerol dehydrogenase, and ascorbate oxidase were purchased from Toyobo USA, Inc. (NY, USA). Water (Optima LC–MS Grade), toluene, sulfuric acid (98%), nitric acid (70%), porcine serum, and phosphate-buffered saline (1×, Gibco PBS, pH 7.2) were purchased from Fisher Scientific (MA, USA). Sliver / silver chloride (Ag / AgCl) ink was purchased from Ercon Incorporated (MA, USA). Glassy carbon electrode (GC, diameter: 3.0 mm) and disc gold electrode (diameter: 1.6 mm) were purchased from Bioanalytical Systems Inc. (IN, USA). Mediator modified screen-printed carbon electrodes (110PHEN) were purchased from Metrohm AG (Switzerland). PILOGEL discs were purchased from ELITechGroup Inc. (UT, USA). Polyethylene terephthalate (PET, 100 μm thick) was purchased from MG Chemicals (BC, Canada). Styrene–ethylene–butylene–styrene block (SEBS, H1062) was purchased from Asahi Kasei (Japan). Double-sided tape (170 μm thick, 9474LE 300 LSE), anisotropic conductive film tape (ACF, 9703), and Tegaderm film were purchased from 3M Science (MN, USA).

[0063] Example Construction of the SWCNT-based NADH sensor and cofactor- integrated biosensor (CIB) 21 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151

[0064] Example biosensors of embodiments (e.g. individual sensors in array 122 of Fig. 1C) were fabricated on gold (Au) electrodes (e.g.134 in Fig.1C) (diameter: 3 mm; 30 nm chromium, Cr / 100 nm Au), deposited and patterned on a PET substrate or SEBS substrate (e.g. 136 in Fig.1C). The SEBS substrate can be fabricated by dissolving SEBS blocks in toluene (150 mg / mL) and vortexed overnight. Then the SEBS solution was drop-cast on the silver trace and cured at 90 °C for 2 hours, followed by the attachment of ACF tape.

[0065] The reference electrode (e.g.138 in Fig.1C) was fabricated by depositing 3.5 μL of Ag / AgCl ink on the Au electrode, then dried on a hot plate at 70 °C for 20 min.

[0066] An acid-treated SWCNT solution (12 mg / mL in PBS) was prepared by dispersing SWCNT into a mixture of sulfuric acid and nitric acid solution (1:3) and heating at 80 °C for 4 hours while stirring followed by centrifuging and washing with PBS to remove the residual acid. For NADH sensors, 3.5 μL of the acid-treated SWCNT solution was drop-cast onto the Au electrode and dried in the ambient environment. For enzymatic CIBs, NAD+was integrated into SWCNT solution (12 mg / mL in PBS) by dispersing acid-treated SWCNT into a NAD+solution (50 mM in PBS) and stirring at 4 °C for 20 hours followed by centrifuging and washing with PBS and water. 3.5 μL of the NAD+integrated SWCNT solution was drop-cast onto the Au electrode and dried in the ambient environment (e.g. forming layer 124 in Fig.1C).

[0067] Then, the electrode was further functionalized with a dehydrogenase layer (DH) by drop-casting an enzyme solution, which was prepared following the instruction provided by the vendor (e.g. forming layer 126 of Fig.1C).

[0068] To develop the BHB sensor, 1.13 μL of the BHB dehydrogenase solution (1056 U / mL) was drop-cast onto the Au / SWCNT electrode and dried in the ambient environment.

[0069] Then, another 1.75 μL of the NAD+integrated SWCNT was drop-cast onto the Au / SWCNT-NAD+ / DH electrode and dried in the ambient environment (e.g. forming layer 128 of Fig.1C). The fabrication of other enzymatic CIBs follows the same procedure described above with the use of their corresponding enzyme solution (800 U / mL for glutamate dehydrogenase and 8000 U / mL for the rest of the enzymes). To eliminate the interference from AA, 1.13 μL of the ascorbic acid oxidase solution (15000 U / mL) was drop-cast onto the Au / SWCNT-NAD+ / DH / SWCNT-NAD+electrode, followed by drop-casting 1 μL of the glutaraldehyde solution (0.4 wt% in PBS solution) (e.g. forming layer 130 of Fig.1C). An anti- 22 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151fouling encapsulation layer was deposited by drop-casting of 1 μL of the PVC solution (0.1 wt% in THF) for 3 times (e.g. forming layer 132 of Fig.1C). Sensors were allowed to dry overnight at 4 °C, while being protected from light. The sensors were stored at 4 °C in the dark when not in use.

[0070] Electrochemical characterization of Example NADH sensors

[0071] Amperometric measurements: the calibration curves were measured in a continuously stirred PBS buffer (1× PBS) at +0 V (vs. Ag / AgCl). All electrochemical characterizations were performed by a potentiostat (CHI 1040C, CH Instruments, Inc.). By stepwise addition of increasing concentrations of the NADH stock solution in the PBS buffer, a calibration plot was obtained. Sensitivity is calculated as the slope divided by the sensor's surface area. The signal degradation of GC, 1,10-phenanthroline-5,6-dione / carbon paste (PD / CP) and SWCNT-based NADH sensor were measured by the percentage of current drop in a 150 μM NADH solution in the PBS buffer after 1-hour amperometric measurement at 0.6 V (vs. Ag / AgCl for GC) or 0 V (vs. Ag / AgCl for PD / CP and SWCNT). The signal-to-noise ratio (SNR) was calculated by the ratio of the sensitivity of analyte to the sensitivity of the interference. The sensitivity of the interference was characterized by stepwise addition of various interferences into the PBS, including metformin, UA, caffeine, APAP, HIS, TRY, and AA.

[0072] Rotating disk electrode linear scan voltammetry (LSV): the electron transfer number and the exchange current density of NADH oxidation by SWCNT were characterized by LSV in 1 mM NADH solution in 1× PBS buffer from +0.4 to +1.0 V (vs. reversible hydrogen electrode, RHE) at a scan rate of 10 mV / s with different rotating speed and were calculated based on the Koutecký–Levich equation.

[0073] Cyclic Voltammetry (CV): to identify the existence of NAD+on SWCNT, the reduction of NAD+was observed at -1.1 V (vs. Ag / AgCl) in the PBS solution at a scan rate of 100 mV / s. The specific area was evaluated by comparing their capacitance obtained from CV measurements conducted in the PBS buffer from −0.2 to +0.2 V (vs. RHE) at different scan rates (10, 25, 50, 75, 100, 125 mV / s).

[0074] Electrochemical characterization of Example CIBs 23 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151

[0075] Amperometric measurements: the sensitivity of BHB, glucose, glutamate, G6P, ethanol, D-lactate, L-leucine, cholesterol, and glycerol was obtained by amperometric measurements as described previously. The sensor selectivity tests were conducted by stepwise addition of different interference molecules into the PBS, including glucose / BHB, lactate, KCl, NaCl, UA, AA. The target analytes were also introduced into the solutions after the interference. The multiplexed sensor test was conducted by stepwise addition of different targets into the porcine serum.

[0076] BHB and glucose sample collection and quantification

[0077] Human sample collection: for sweat samples, a subject’s inner wrist was first cleaned with deionized water and ethanol, followed by sweat gland stimulation by iontophoresis for 5 min (Macroduct sweat collection system, ELITechGroup Inc.). Then, a sweat collector was attached to the sweating region for 30 min. For whole saliva samples, the collection was performed by using a passive drool method (Saliva Collection Aid, Salimetrics).

[0078] Capillary blood measurement: a commercially-available meter (Precision Xtra, Abbott) with BHB and glucose test strips (Abbott) were used to quantify BHB or glucose in capillary blood sampled by fingerprick before saliva and sweat sample collection from human subjects.

[0079] Fluorometric measurement: BHB and glucose levels from undiluted human sweat and saliva samples, and mice serum samples were quantified with assays (ab180876 and ab65333) performed with a fluorescent microplate reader (Omega, BMG LABTECH).

[0080] Characterization of Example chemical compositions

[0081] Scanning transmission electron microscope (S / TEM): the chemical composition of SWCNT after NAD+integration was characterized by energy dispersive spectroscopy (EDS) (Oxford X-MaxTEM 100N TLE Windowless SDD 100 mm2) in a Titan S / TEM (FEI). The S / TEM sample was prepared by dissolving NAD+integrated SWCNT in ethanol then drop- casting onto a support film grid (Ultrathin Carbon Film on Lacey Carbon Support Film, 400 mesh, Copper, TED PELLA, Inc., CA). 24 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151

[0082] In-vitro biocompatibility test

[0083] Cell culture assay: the cell toxicity was evaluated using human foreskin fibroblasts (HFFs). Fabricated devices or components were sterilized (UV for 15 mins) before being incubated in the sterile Dulbecco's Modified Eagle Medium (DMEM) for 7 days at 37 °C to obtain a “conditioned medium”. HFFs were cultured in the conditioned medium (10% of fetal bovine serum was added) for 24 hours. The positive control group was created by the addition of AgNO3into the DMEM to reach a concentration of 10 μg / mL. To quantify the viability, the cells were analyzed by PrestoBlue™ Cell Viability Reagent (Invitrogen) using the fluorescence (plate- reader SynergyMX, Biotech) with excitation wavelength 560 nm and emission wavelength 590 nm. Each study involved two biological replicates and two technical replicates.

[0084] NADH electrochemical reaction model

[0085] A two-dimensional microkinetic model in an aqueous solution was constructed and simulated using COMSOL Multiphysics (Ver.5.5). The model included two layers: the electrode layer and the diffusion layer. A constant potential (E_s0) was applied to the electrode’s surface. The reaction of electrochemical oxidation at the electrode’s surface proceeded as

[0086] The kinetic rate of electrochemical reaction was described by the concentration Butler-Volmer equation

[0087] where i is the local current density of NADH oxidation at a given location on the porous electrode; i0is the exchange current density, determined by analysis of electrochemical data; cNADHand cNADare local concentrations of NADH and NAD+; cNADH,0and cNAD,0are the concentrations of NADH and NAD+in bulk solution; αa = αc = 0.5 is the transfer coefficient; F is the Faraday constant; R is the gas constant; T is the temperature; η is over potential, defined as the difference between the applied potential and the standard redox potential of NAD+ / NADH. 25 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151

[0088] Governing differential equations:

[0089]

[0090] diffusion layer (Diff) was defined as the distance between the boundary of the diffusion layer and the boundary of the enzyme layer. A parametric sweep of the diffusion layer length was done to investigate the effect of the diffusion.

[0091] Enzymatic electrochemical reaction hybrid model

[0092] The model included three layers: the electrode layer, the enzyme layer, and the diffusion layer. The electrode layer was where the electrochemical reaction happened, and it consisted of carbon nanotubes (CNT). The width (CNT_w) of the electrode layer was measured by experiments and the thickness (CNT_h) of the electrode was estimated from the density of the materials used. A layer of enzyme was deposited on the top of the electrode and the enzymatic reaction happened within the enzyme layer. The width of the enzyme layer was considered to be the same as the electrode layer, and the thickness (Elayer_h) was estimated from the density of the materials used. Outside the enzyme layer is the diffusion layer. The length of the diffusion layer (Diff) was defined as the distance between the boundary of the diffusion layer and the boundary of the enzyme layer. The boundary condition at the diffusion layer was considered to be equilibrated with the bulk solution.

[0093] A porous electrode module was used to simulate the electrode layer. The specific area (e.g., area_CNT) and the electrode volume fraction (f_s) were obtained by calculations based on the electrode materials’ specification sheets. A constant potential (E_s0) was applied to 26 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151the electrode’s surface. The reaction of electrochemical oxidation at the electrode’s surface proceeded as

[0094] reaction was described by the concentration Butler-Volmer equation

[0095] at a given location on the porous electrode; i0 is the exchange current density, determined by analysis of electrochemical data; cNADHand cNADare local concentrations of NADH and NAD+; cNADH,0and cNAD,0are the concentrations of NADH and NAD+in bulk solution; αa=αc=0.5 is the transfer coefficient; F is the Faraday constant; R is the gas constant; T is the temperature; η is over potential, defined as the difference between the applied potential and the standard redox potential of NAD+ / NADH.

[0096] A porous matrix module was used to simulate the enzymatic layer. The enzymatic reaction proceeded as

[0097] The kinetic rate of the enzymatic reaction was described by Michaelis-Menten equation:

[0098] where rPis the generation rate of the product; rSis the consumption rate of the substrate; kcatis the catalytic rate constant of the enzymatic reaction; KMis the Michaelis constant 27 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151of the enzyme; cE,0is the initial concentration of enzyme; cBHBis the local concentration of β- hydroxybutyrate.

[0099] Governing differential equations:

[0100] cBHB, cacac, cNADH, cNAD are the local concentrations of β- hydroxybutyrate, acetoacetate, NADH and NAD+.

[0101] At the enzyme layer boundary

[0102] where cNADH,0 and cNAD,0 are the concentration of NADH and NAD+in bulk solution.

[0103] At the diffusion layer boundary

[0104] where cBHB,0and cacac,0are the concentration of β-hydroxybutyrate and acetoacetate in bulk solution.

[0105] A total current of electrochemical reaction was calculated to evaluate the performance of the hybrid system. A parametric sweep of the specific area of porous electrode and the exchange current density was done to investigate the effect of the catalysts’ properties. A 28 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151parametric sweep of the catalytic rate constant (kcat) and the Michaelis constant (KM) was also done to demonstrate and guide the potential application on different enzymes.

[0106] In-vivo CIB array characterization in mice

[0107] Animals: mixed gender, body weight 20-28 g, 3-to-4-month-old C57BL / 6J (C57BL / 6J, Catalog No: 000664, The Jackson Laboratory, ME, USA) mice were used in this study. All animal studies were performed according to the protocols approved by the University of California, Los Angeles Animal Research Committee, under the ARC protocol number 2019- 019. The experimental sample size (8 mice in total) was determined by a similar study. The methods were carried out in accordance with the relevant guidelines and regulations in full compliance with the ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines 2.0.

[0108] In-vivo test in mice: before each study, animals were weighed to make sure that their body weights were over 20 g. Mice were anesthetized by isoflurane via inhalation (Isoflurane vaporizer, Somni Inc.). The induction of the anesthesia was done with 3-5% isoflurane no longer than 2 min and the maintenance of the anesthesia was done with 0.5-2% isoflurane. Toe pinch was used to validate the state of anesthesia every 15 min throughout the entire procedure. A pneumotach (biopac) was connected to the mouthpiece of the anesthetic system to monitor the airway pressure change. Once the animal was fully anesthetized, one incision was made to the skin of the mid-thoracic level for blood collection and one incision was made to the skin of the base of tail for subcutaneous placement of the recording sensors. The pre- baseline blood sample was collected, followed by the subcutaneous placement of the sensors. One sensor array (e.g.122 in Fig.1C) with two types of sensors (BHB and glucose), one reference electrode and one control electrode will be placed topically near the abductor caudae dorsalis muscle close to the base of the tail. The skin will cover and stabilize the sensor arrays and biocompatible Tegaderm tape (3M) was used to further stabilize the sensor at the recording sites. After being stabilized, the sensor arrays were connected to the potentiostat, and the signals were collected at a sampling rate of 10 Hz.

[0109] The recording session had two phases: the pre-nutrient delivery phase and the nutrient delivery phase. After the blood collection and sensor placement, the sensors were 29 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151stabilized subcutaneously to reach the equilibrium status in-vivo and establish the pre-nutrient delivery baseline. After the pre-nutrient delivery baseline was established, one dose of BHB (2.5 g / kg in saline solution) or one dose of glucose (1.0 g / kg in saline solution) was given to the animal via tail-vein injections. After around 20 min of recording, another dose of BHB (2.5 g / kg in saline solution) was given to the animal in a similar way. The recording was stopped 20 min after the second dose and the post-nutrient delivery blood was collected.

[0110] Surveys for oxidoreductase library and measurable biomarkers

[0111] Enzyme data source: the BRENDA (The Comprehensive Enzyme Information System) database (A. Chang, L. Jeske, S. Ulbrich, J. Hofmann, J. Koblitz, I. Schomburg, M. Neumann-Schaal, D. Jahn, D. Schomburg, BRENDA, the ELIXIR core data resource in 2021: new developments and updates. Nucleic Acids Res.49, 498–508 (2021)) served as the resource for estimating the total number of oxidoreductases. Focusing on enzymes categorized under the EC number 1, specifically those identified as oxidoreductases, the search criteria included enzymes with names containing peroxidase, reductase, dehydrogenase, oxidase, oxygenase, and hydroxylase.

[0112] Categorization of enzymatic reactions and measurable biomarkers: enzymatic reactions producing hydrogen peroxide, H2O2, were labeled as non-cofactor-based catalytic reactions, while the remaining reactions involving NAD+or other cofactors were considered cofactor-based catalytic reactions. The analyte of enzymatic reaction containing NAD+or oxygen, O2, was counted as a measurable biomarker.

[0113] Wireless printed circuit board (PCB) module

[0114] A wireless PCB module can be used to drive the sensing circuits, and to process and communicate the obtained data wirelessly upon user commands. This circuit consists of several layers to enclose the sweat collection module and interface it with sensing modules. The main PCB section encapsulates a flat flexible cable connection to interface with the sensor (e.g. array 122 in Fig.1C). Utilizing an anisotropic conductive film, the flexible cable was connected to the fabricated flexible electrodes. 30 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151

[0115] After the iontophoresis process, the sensing mode is activated. At this stage, the microcontroller will activate the LMP91000 potentiostat chip (Texas Instruments) through I2C- controlled signals. This chip is programmed to apply 0 V across the working and reference electrodes connected to the sensor. Then, to filter out high-frequency interference and the user's motion artifacts, the output voltage of the potentiostat's internal transimpedance amplifier is passed through a fifth-order low-pass filter (LPF). The LPF was applied using a MAX7422 chip (Maxim Integrated) with a cut-off frequency of 1 Hz. Through the microcontroller's built-in 12- bit analog-to-digital (ADC) unit then converts the processed analog output of the LPF into the digital domain for real time biomarker monitoring which is then plotted on the LCD (liquid crystal display, st7735-TFT-LCD, Sitronix Technology Corporation) and transfers this data to the user's smartphone.

[0116] To power the PCB, a single miniaturized rechargeable lithium-ion polymer battery with a nominal voltage of 3.7 V was used. Additionally, in this design, low / ultralow power components were used to make the device wearable and feasible for on-body testing. For a specific application and duration of tests, it was characterized that peak supply current levels on the order of 100 mA must be drawn from the battery. This requirement can vary based on different modes and durations of applications. 31 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151Table 2. Physiological range of various analytes in blood and saliva Analyte Blood concentration Saliva concentration Reference BHB 0.1 – 6.3 mM 0 – 360 μM This work Glucose 4 – 17 mM 0.03 – 1 mM (1) L-glutamate 60 – 80 μM 18 – 49 μM (2) G6P 100 – 300 μM (intracellular) Not reported (3) Ethanol 10 – 70 mM 10 – 60 μM (4,5) D-lactate 30 – 180 μM Not reported (6) L-leucine 150 – 500 μM 10 – 300 μM (7) Cholesterol 4.8 – 5.4 mM 0.02 – 5.46 μM (8,9) Glycerol 55 – 228 μM Not reported (10)

[0117] The following are the references for the above Table 2:

[0118] 1. P. Abikshyeet, V. Ramesh, N. Oza, Glucose estimation in the salivary secretion of diabetes mellitus patients. Diabetes Metab. Syndr. Obes.5, 149–154 (2012).

[0119] 2. W. Bai, W.-L. Zhu, Y.-L. Ning, P. Li, Y. Zhao, N. Yang, X. Chen, Y.-L. Jiang, W.-Q. Yang, D.-P. Jiang, L.-Y. Chen, Y.-G. Zhou, Dramatic increases in blood glutamate concentrations are closely related to traumatic brain injury-induced acute lung injury. Sci. Rep. 7, 5380 (2017).

[0120] 3. A. Scinska-Bienkowska, E. Wrobel, D. Turzynska, A. Bidzinski, E. Jezewska, H. Sienkiewicz-Jarosz, K. Golembiowska, W. Kostowski, A. Kukwa, A. Plaznik, P. 32 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151Bienkowski, Glutamate concentration in whole saliva and taste responses to monosodium glutamate in humans. Nutr. Neurosci. (2006).

[0121] 4. A. Zhu, R. Romero, H. R. Petty, An enzymatic fluorimetric assay for glucose-6-phosphate: application in an in vitro Warburg-like effect. Anal. Biochem.388, 97–101 (2009).

[0122] 5. K. E. L. McColl, B. Whiting, M. R. Moore, A. Goldberg, Correlation of ethanol concentrations in blood and saliva. Clin. Sci.56, 283–286 (1979).

[0123] 6. C. W. Ludvigsen, J. R. Thurn, G. L. Pierpont, J. H. Eckfeldt, Kinetic enzymic assay for D(-)-lactate, with use of a centrifugal analyzer. Clin. Chem.29, 1823–1825 (1983).

[0124] 7. N. Yoshii, K. Sato, R. Ogasawara, Y. Nishimura, Y. Shinohara, S. Fujita, Effect of mixed meal and leucine intake on plasma amino acid concentrations in young men. Nutrients 10, 1543 (2018).

[0125] 8. A. M. Casas-Ferreira, M. del Nogal-Sánchez, E. Rodríguez-Gonzalo, B. Moreno-Cordero, J. L. Pérez-Pavón, Determination of leucine and isoleucine / allo-isoleucine by electrospray ionization-tandem mass spectrometry and partial least square regression: Application to saliva samples. Talanta 216, 120811 (2020).

[0126] 9. S. Karjalainen, L. Sewón, E. Soderling, B. Larsson, I. Johansson, O. Simell, H. Lapinleimu, R. Seppänen, Salivary cholesterol of healthy adults in relation to serum cholesterol concentration and oral health. J. Dent. Res.76, 1637–1643 (1997).

[0127] 10. J. Lebeck, B. Brock, Plasma glycerol levels in men with hypertriglyceridemia. Scand. J. Clin. Lab. Invest. (2021).

[0128] The herein described subject matter sometimes illustrates different components contained within, or connected with, different other components. It is to be understood that such depicted architectures are illustrative, and that in fact many other architectures can be implemented which achieve the same functionality. In a conceptual sense, any arrangement of components to achieve the same functionality is effectively "associated" such that the desired functionality is achieved. Hence, any two components herein combined to achieve a particular functionality can be seen as "associated with" each other such that the desired functionality is 33 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151achieved, irrespective of architectures or intermedial components. Likewise, any two components so associated can also be viewed as being "operably connected," or "operably coupled," to each other to achieve the desired functionality, and any two components capable of being so associated can also be viewed as being "operably coupleable," to each other to achieve the desired functionality. Specific examples of operably coupleable include but are not limited to physically mateable and / or physically interacting components and / or wirelessly interactable and / or wirelessly interacting components and / or logically interacting and / or logically interactable components.

[0129] With respect to the use of plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0130] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

[0131] Although the figures and description may illustrate a specific order of method steps, the order of such steps may differ from what is depicted and described, unless specified differently above. Also, two or more steps may be performed concurrently or with partial concurrence, unless specified differently above. Such variation may depend, for example, on the software and hardware systems chosen and on designer choice. All such variations are within the scope of the disclosure. Likewise, software implementations of the described methods could be accomplished with standard programming techniques with rule-based logic and other logic to accomplish the various connection steps, processing steps, comparison steps, and decision steps.

[0132] It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation, no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases 34 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and / or "an" should typically be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, typically means at least two recitations, or two or more recitations).

[0133] Furthermore, in those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to "at least one of A, B, or C, etc." is used, in general, such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0134] Further, unless otherwise noted, the use of the words “approximate,” “about,” “around,” “substantially,” etc., mean plus or minus ten percent.

[0135] Although the present embodiments have been particularly described with reference to preferred examples thereof, it should be readily apparent to those of ordinary skill in the art that changes and modifications in the form and details may be made without departing 35 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151from the spirit and scope of the present disclosure. It is intended that the appended claims encompass such changes and modifications. 36 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151

Claims

WHAT IS CLAIMED IS:

1. A device comprising: a cofactor-integrated biosensor including a single-wall-carbon-nanotubes (SWCNT) framework.

2. The device of claim 1, wherein the biosensor is configured to simultaneously adsorb cofactors, self-mediate cofactor redox reactions, and enhance reaction rates at the limit of enzyme activity.

3. The device of claim 1, wherein the framework includes a plurality of functional layers, including one or more enzyme layers for detection and supplemental interference elimination, and an encapsulation layer for stabilization and anti-fouling.

4. The device of claim 3, wherein the enzyme layers are configured to respectively catalyze target-induced reactions and consume the dominant interference sources with no reaction crosstalk.

5. The device of claim 1, wherein the framework includes an electrode with a high specific area configured to increase loading of cofactor molecules and detection / interference-elimination enzymes for enhanced signal-to-noise ratio (SNR) measurements.

6. The device of claim 1, wherein the biosensor includes an electrode formed on a substrate.

7. The device of claim 6, wherein the electrode comprises gold.

8. The device of claim 6, wherein the framework includes a first functional layer comprising an acid-treated SWNCT solution cast onto the electrode. 37 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-11519. The device of claim 8, wherein the SWNCT solution includes nicotinamide adenine dinucleotide (NAD+).

10. The device of claim 7 or 8, wherein the framework further comprises an enzymatic layer.

11. The device of claim 10, wherein the enzymatic layer includes a BHB dehydrogenase solution.

12. The device of claim 10, wherein the framework further comprises a second functional layer comprising nicotinamide adenine dinucleotide (NAD+) integrated SWCNT disposed on the enzymatic layer.

13. The device of claim 12, wherein the framework further comprises an interference elimination layer comprising an ascorbic acid oxidase solution disposed on the second functional layer.

14. The device of claim 12 or 13, wherein the framework further comprises an anti-fouling encapsulation layer comprising a PVC solution.

15. The device of claim 1, wherein the biosensor includes a plurality of compartments for analyzing biofluids, each of the compartments comprising the SWCNT framework and targeting a respective cofactor. 38 2024-134-PCT S. Emaminejad et al. Atty. Dkt.102352-1151

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