Amino acid sensor system for health monitoring

Abstract

Methods, apparatus, and systems for determining whether a subject is experiencing, has experienced, or is at risk of experiencing at least one of cardiovascular function, NT-proBNP, BNP, Cardiac Myosin-Binding Protein C (cMyBP-C), Mid-region pro-atrial natriuretic peptide (MR-proANP), inflammation, a cytokine (such as IL-6 or TNF-α), C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen, including monitoring the amount or concentration of at least one amino acid in a subject, wherein the at least one amino acid is an amino acid that correlates to a cardiovascular issue; detecting a deviation in the amount or concentration of the at least one amino acid away from a baseline value of amount or concentration of the at least one amino acid; measuring a change in the amount or concentration of the at least one amino acid after detecting the deviation away from the baseline value of amount or concentration of the at least one amino acid; and determining whether the subject is experiencing, has experienced, or is at risk of experiencing a changing (e.g., worsening or improving) condition or health status based on the measured change of the amount or concentration of the at least one amino acid.

Description

AMINO ACID SENSOR SYSTEM FOR HEALTH MONITORING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of the filing date of, U.S. Provisional Application No. 63 / 754,911, filed on February 6, 2025, the disclosure of which is hereby incorporated by reference herein in its entirety.INCORPORATION BY REFERENCE STATEMENT

[0002] The Sequence Listing XML file, identified as “UOC-25041 WO Sequence Listing,” having a file size of 6 KB and created on February 3, 2026, is incorporated by reference in its entirety as part of this application.FIELD OF THE INVENTION

[0003] This invention relates generally to an amino acid sensor system for health monitoring, including but not limited to continuous sensing of phenylalanine for heart failure monitoring.BACKGROUND OF THE INVENTION

[0004] This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and / or claimed below.This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0005] Biochemical monitors have witnessed significant commercial success in the form of continuous glucose monitors for diabetes management. Emerging technologies are pursuing biochemical monitoring of conditions such as cardiovascular disease. For example, NT-proBNP monitoring is being developed for heart failure monitoring, using technologies such as electrochemical aptamer sensors to measure NT-proBNP in interstitial fluid. For most biochemical monitoring applications, accuracy is a critical performance parameter. For NT-proBNP, monitoring accuracy may be limited by blood to interstitial fluid correlation, or by the electrochemical sensor itself, or other factors. Therefore, alternate measures, or adjunct measures, may be merited. What is needed are additional methods to measure various health issues, such as heart failure status or to replace or improve the correlation of NT-proBNP, BNP, or other measures to blood concentrations. Such measures could improve the standard of care in disease management such as in heart failure or other conditions, or for general health and wellness applications.SUMMARY OF THE INVENTION

[0006] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0007] Many of the drawbacks and limitations stated above can be resolved by creating novel and advanced interplays of chemicals, materials, sensors, electronics, microfluidics, algorithms, computing, software, systems, and other features or designs, in a manner that affordably, effectively, conveniently, intelligently, or reliably brings sensing technology into proximity with biofluid and analytes.

[0008] As described above in the Background section, there are currently issues with developing monitors for health issues, such as issues of the heart (such as monitors for markers that can be indicative of such issues, e.g., cardiovascular disease). For example, NT-proBNP or BNP monitoring is being developed for heart failure monitoring, using technologies such as electrochemical aptamer sensors to measure NT-proBNP or BNP in interstitial fluid. However, accuracy in NT-proBNP or BNP monitoring may be limited by blood to interstitial fluid correlation, or by the electrochemical sensor itself, or other factors. Sensors such as aptamer sensors for highly dilute biomarkers such as IL-6, CRP, and NT-proBNP also typically require hydrophobic modifications to aptamers to drive strong binding interactions, which can increase the probability that other hydrophobic molecules in the body can interfere with a sensor. And, for most biochemical monitoring applications, accuracy is a critical performance parameter (without accuracy, the use of the data to accurately predict cardiovascular issues, or risk of same, is diminished or eliminated altogether). As a result, such sensors are not yet used for NT-proBNP monitoring.

[0009] However, it has recently been shown that certain amino acids may be biomarkers for various diseases, disorders, and conditions covering a host of health issues - including cardiovascular issues. Unfortunately, recent demonstrations of this (relative to heart conditions) are based on single-data point measures of an amino acid via a blood draw, and in some cases, ratios of amino acids to one another are required to correct for confounding effects such as diet, kidney function, and other factors. As such, the use of these amino acids for possible correlation to cardiovascular issues suffers the problems of being invasive, inaccurate, time-consuming, requiring scheduling and travel of the subject (such as to a clinic or medical facility for blood draw).

[0010] Aspects of the present invention, however, overcome the drawbacks described above by providing methods, apparatus, and systems for monitoring the amount of concentration of at leastone amino acid from a subject (e.g., in a sample fluid such as interstitial fluid), including continuous monitoring, which results in multiple data points being taken over a period of time. By these methods, apparatus, and systems, aspects of the present invention provide continuous or periodic measurements of the at least one amino acid (as opposed to the single data point measurements seen in current methods and apparatus) and provide same in a more readily accessed fluid (e.g., interstitial fluid) that does not require the same level of invasiveness, time consumption, etc. as currently seen with blood draws.

[0011] One aspect of the present invention is directed to a method for determining whether a subject is experiencing, has experienced, or is at risk of experiencing a health issue (such as a worsening condition or health status). The method includes the steps of (1) monitoring the amount or concentration of at least one amino acid in a subject, wherein the at least one amino acid is an amino acid that correlates to at least one of cardiovascular function, NT-proBNP, BNP, Cardiac Myosin-Binding Protein C (cMyBP-C), Mid-region pro-atrial natriuretic peptide (MR-proANP), inflammation, a cytokine (such as IL-6 or TNF-a), C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen; (2) detecting a deviation in the amount or concentration of the at least one amino acid away from a normal or a baseline value of amount or concentration of the at least one amino acid; (3) measuring a change in the amount or concentration of the at least one amino acid after detecting the deviation away from the baseline value of amount or concentration of the at least one amino acid; and (4) determining whether the subject is experiencing, has experienced, or is at risk of experiencing a changing (e.g., worsening or improving) condition or health status based on the measured change of the amount or concentration of the at least one amino acid.

[0012] Another aspect of the present invention is directed to a method of determining whether a subject is experiencing, has experienced, or is at risk of experiencing a health issue (such as a worsening condition or health status). The method includes the steps of (1) continuously monitoring at least one amino acid in a subject via a plurality of measurements of the amount or concentration of the at least one analyte in the subject, wherein the at least one amino acid is an amino acid that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine, C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen; (2) detecting a change in the amount or concentration of the at least one amino acid in the subject over the plurality of measurements, the change being an increase or a decrease in the amount or concentration of the at least one analyte; and (3) determining whether the subject is experiencing, has experienced, or is at risk of experiencing a changing (e.g., worsening or improving) condition or health status based on the change of the amount or concentration of the at least one amino acid.[0013| Yet another aspect of the present invention is directed to a method of determining whether a subject is experiencing, has experienced, or is at risk of experiencing a health issue (such as a worsening condition or health status). The method includes the steps of (1) monitoring the amount or concentration of at least two amino acids in a subject, wherein the at least two amino acids have a ratio that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine, C-reactive protein, kidney function, creatinine cystatin-C, blood-urea-nitrogen; (2) detecting a deviation in the ratio away from a baseline ratio; and (3) measuring a change in the ratio after detecting the ratio; and (4) determining whether the subject is experiencing, has experienced, or is at risk of experiencing at least one of a changing (e.g., worsening or improving) condition or health status based on the measured change in the ratio.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:

[0011] FIGS. 1A-C are a series of graphs showing the prognostic value of phenylalanine (PHE) and B-type natriuretic peptide (BNP), with FIG. 1 A showing the Kaplan-Meier curves for patients with PIIE > 65.6 pM versus PIIE < 65.6 pM; FIG. IB showing the Kaplan-Meier curves for patients with BNP > 510 pg / mL versus BNP < 510 pg / mL; and FIG. 1C showing the Kaplan-Meier curves for patients of four subgroups based the levels of PHE and BNP [from Yeh J-K, et al.. Prognostic Significance of Phenylalanine in Heart Failure: Clinical Insights and Inter-Organ Crosstalk Snapshot. J. of Clin. Med. 2024; 13(14):4251. https: / / doi.org / 10.3390 / jcml3144251],

[0012] FIG. 2 is a graph showing prognostic value of phenylalanine and leucine amino acids (PLA) by showing the receiver operating characteristic (ROC) curves for PLA, APACHE II, and NUTRIC scores [from Tsou Y-L, et al., Combining Phenylalanine and Leucine Levels Predicts 30-Day Mortality in Critically III Patients Better than Traditional Risk Factors with Multicenter Validation. Nutrients. 2023; 15(3):649. https: / / doi.org / 10.3390 / nul5030649],

[0013] FIGS. 3A-B include two graphs demonstrating the prognostic value of phenylalanine / leucine ratios in patients with heart failure [from Hiraiwa H, et al., Prognostic value of leucine / phenylalanine ratio as an amino acid profile of heart failure. Heart Vessels. 2021 Jul;36(7):965-977. doi: 10.1007 / s00380-020-01765-z. Epub 2021 Jan 22. PMID: 33481086],

[0014] FIGS. 4A-E are a series of graphs depicting the prognostic value of phenotype and genotype [from Wang, et al., Stress Hyperphenylalaninemia Is Associated With Mortality in Cardiac ICU: Clinical Factors, Genetic Variants, and Pteridines. Critical Care Medicine 50(1 l):p 1577-1587, November 2022; DOI: 10.1097 / CCM.0000000000005640].[0015| FIG. 5 is a schematic of an example of a sensor device including a plurality of microneedle sensors.

[0016] FIG. 6 is a schematic of another example of a sensor device including a single microneedle or needle or microstrip sensor.

[0017] FIG. 7 is a schematic showing a working electrode for an example of a sensor device having a plurality of aptamers bound thereto, and illustrating examples of aptamer conformations with and without analyte bound to the aptamer.

[0018] FIG. 8 is a diagram of an example environment in which systems and / or methods described herein may be implemented.

[0019] FIG. 9 is a schematic showing an example wearable monitoring device according to the present invention.DEFINITIONS

[0020] As used herein, “continuous sensing’’ with a “continuous sensor’’ means a sensor that changes in response to changing concentration of at least one solute in a solution such as an analyte. Similarly, as used herein, “continuous monitoring” means the capability of a device to provide multiple measurements of an analyte over time.

[0021] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration or percentage is meant to encompass variations of ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments from the specified amount, as such variations are appropriate to perform the disclosed method.

[0022] As used herein, the term “electrode” means any material that is electrically conductive such as gold, platinum, nickel, silicon, conductive liquid infused materials such as ionic liquids, PEDOT:PSS, conductive oxides, carbon, boron-doped diamond, nanotubes or nanowire meshes, or other suitable electrically conducting materials.

[0023] As used herein, the term “blocking layer” means a homogeneous or heterogeneous layer of material or of one or more types of molecules on an electrode which reduce electrochemical background current and / or current due to electrochemical interference, and which may promote proper freedom of movement for the aptamer which is required for creating a measurable response to analyte concentration.

[0024] As used herein, the term “antifouling layer” means a homogeneous or heterogeneous layer of material or of one or more types of molecules on a surface which reduces fouling on a surface compared to if such an antifouling layer was not utilized.[0025| As used herein, the term “aptamer” means a molecule that undergoes a conformation or binding change as an analyte binds to the molecule, and which satisfies the general operating principles of the sensing method as described herein. Such molecules are, e.g., natural or modified DNA, RNA, or XNA oligonucleotide sequences, spiegelmers, peptide aptamers, and affimers and other affinity-based probes. Modifications may include substituting unnatural nucleic acid bases for natural bases within the aptamer sequence, replacing natural sequences with unnatural sequences, or other suitable modifications that improve sensor function, but which behave analogous to traditional aptamers. Two or more aptamers bound together can also be referred to as an aptamer (i.e., not separated in solution). Aptamers can have molecular weights of at least 1 kDa, 10 kDa, or 100 kDa.

[0026] As used herein, the term “redox tag” or “redox molecule” means any species such as small or large molecules with a redox active portion that when brought adjacent to an electrode can reversibly transfer at least one electron with the electrode. Redox tag or molecule examples include methylene blue, ferrocene, quinones, or other suitable species that satisfy the definition of a redox tag or molecule. In some cases, a redox tag or molecule is referred to as a redox mediator. Redox tags or molecules may also exchange electrons or change in behavior when brought into proximity with other redox tags or molecules. Exogenous redox molecules are those added to a device, e.g., they are not endogeneous and provided by the sample fluid to be tested.

[0027] As used herein, the term “change in electron transfer” means a redox molecule whose electron transfer with an electrode has changed in a measurable manner. This change in electron transfer can, for example, originate from availability for electron transfer, distance from an electrode, diffusion rate to or from an electrode, a shift or increase or decrease in electrochemical activity of the redox molecule, or any other embodiment as taught herein that results in a measurable change in electron transfer between the redox molecule and the electrode.

[0028] As used herein, the term “sensing monolayer” means at least a plurality of aptamers on a working electrode, which may also include a plurality of molecules or mixtures of molecules that form a blocking layer or an anti-fouling layer.

[0029] As used herein, the term “analyte” means any solute in a solution or fluid which can be measured using a sensor. Analytes can be small molecules, proteins, peptides, electrolytes, acids, bases, antibodies, molecules with small molecules bound to them, DNA, RNA, drugs, chemicals, pollutants, or other solutes in a solution or fluid.

[0030] As used herein, the term “correlative element” means any data, equation, algorithm, software, or other stored information that will utilize a first biochemical measurement for the prediction of a second biochemical measurement or prediction of health or disease status.DETAILED DESCRIPTION OF THE INVENTION

[0031] One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0032] Certain embodiments of the disclosed invention show sensors as simple individual elements. It is understood that many sensors require two or more electrodes, reference electrodes, or additional supporting technology or features which are not captured in the description herein. Sensors can be in duplicate, triplicate, or more, to provide improved data and readings. Sensors may provide continuous or discrete data and / or readings. Certain embodiments of the disclosed invention show sub-components of what would be sensing devices with more sub-components needed for use of the device in various applications, which are known (e.g., a reference or counter electrode, a battery, antenna, adhesive, an electronic display), and for purposes of brevity and focus on inventive aspects, such components may not be explicitly shown in the diagrams or described in the embodiments of the disclosed invention. All ranges of parameters disclosed herein include the endpoints of the ranges.

[0033] As described above in the Background section, there are currently issues with developing monitors for issues of the heart (such as monitors for markers that can be indicative of such issues, e.g., cardiovascular disease). For example, NT-proBNP or BNP monitoring is being developed for heart failure monitoring, using technologies such as electrochemical aptamer sensors to measure NT-proBNP or BNP in interstitial fluid. However, accuracy in NT-proBNP or BNP monitoring may be limited by blood to interstitial fluid correlation, or by the electrochemical sensor itself, or other factors. And, for most biochemical monitoring applications, accuracy is a critical performance parameter (without accuracy, the use of the data to accurately predict cardiovascular issues, or risk of same, is diminished or eliminated altogether). As a result, such sensors are not yet used for NT-proBNP monitoring.

[0034] However, it has recently been shown that certain amino acids may be biomarkers for various health issues. For example, FIGS. 1 A-C demonstrate that phenylalanine (Phe) may be as sensitive as BNP for predicting accumulative survival in heart failure. FIG. 2 demonstrates that multiple amino acids and / or their ratios can predict all-cause mortality. FIGS. 3A-Bdemonstrates both the sensitivity and specificity of Phe to Leucine ratio for heart failure survival. And FIGS. 4A-E demonstrates that Phe predicts accumulative survival in the intensive care unit.

[0035] More specifically, FIGS. 1A-C are a series of three graphs showing the prognostic value of phenylalanine (PHE) and B-type natriuretic peptide (BNP), with FIG. 1A showing the Kaplan-Meier curves for patients with PHE > 65.6 pM versus PHE < 65.6 pM; FIG. IB showing the Kaplan-Meier curves for patients with BNP > 510 pg / mL versus BNP < 510 pg / mL; and FIG. 1C showing the Kaplan-Meier curves for patients of four subgroups based the levels of PHE and BNP [from Yeh J-K, et al.. Prognostic Significance of Phenylalanine in Heart Failure: Clinical Insights and Inter-Organ Crosstalk Snapshot. J. of Clin. Med. 2024; 13(14):4251. https: / / doi.org / 10.3390 / jcml3144251]. The results showed that elevated pre-discharge Phe levels (>65.6 pM) in patients with heart failure with reduced ejection fraction (HFrEF) were associated with a higher risk of all-cause mortality over a median follow-up of 4.5 years [Yeh et al.]. BNP, NT-proBNP, cMyBP-C (Cardiac Myosin-Binding Protein C), MR-proANP (Midregion pro-atrial natriuretic peptide), and other analytes associated with cardiovascular stress such as heart failure correlate with each other and therefore correlate to Phe.

[0036] FIG. 2 is a graph showing prognostic value of phenylalanine leucine amino acid (PLA) by showing the receiver operating characteristic (ROC) curves for PLA, APACHE II, and NUTRIC scores [from Tsou Y-L, et al., Combining Phenylalanine and Leucine Levels Predicts 30-Day Mortality in Critically III Patients Better than Traditional Risk Factors with Multicenter Validation. Nutrients. 2023; 15(3):649. https: / / doi.org / 10.3390 / nul5030649]. The APACHE II score measures the severity of disease and is used to predict the risk of death for ICU patients, and the NUTRIC score measures the nutritional risk of critically ill patients in an ICU. The Tsou study examined combined levels of phenylalanine and leucine amino acids (PLA), and determined that PLA scores predicted 30-day mortality better than APACHE II and NUTRIC scores [Tsou, et al.].

[0037] FIGS. 3A-B includes two graphs demonstrating the prognostic value of phenylalanine / leucine ratios in patients with heart failure [from Hiraiwa H, et al., Prognostic value of leucine / phenylalanine ratio as an amino acid profile of heart failure. Heart Vessels. 2021 Jul;36(7):965-977. doi: 10.1007 / s00380-020-01765-z. Epub 2021 Jan 22. PMID:33481086], Plasma amino acid levels were measured when the patients were stabilized at discharge. Cardiac events were defined as a composite of cardiac death and hospitalization for worsening heart failure (HF). A total of 46 cardiac events occurred during the median follow-up period of 238 (interquartile range 93-365) days. The median leucine / phenylalanine ratio was significantly lower in patients with cardiac events than in those without cardiac events (1.4 vs.1.8, P < 0.001). The best cutoff value of the leucine / phenylalanine ratio was determined as 1.7 inthe receiver operating characteristic (ROC) curve for cardiac events. Following a Kaplan-Meier survival analysis, the low group (leucine / phenylalanine ratio < 1.7, n = 72) had more cardiac events than the high group (leucine / phenylalanine ratio > 1.7, n = 85) (log-rank, P < 0.001). Multivariate Cox proportional hazards regression analysis showed that the leucine / phenylalanine ratio was an independent predictor of cardiac events. [See Hiraiwa et al.]

[0038] FIGS. 4A-E are a series of graphs showing the prognostic value of phenotype and genotype [from Wang, et al., Stress Hyperphenylalaninemia Is Associated With Mortality in Cardiac ICU: Clinical Factors, Genetic Variants, and Pteridines. Critical Care Medicine 50(1 l):p 1577-1587, November 2022; DOI: 10.1097 / CCM.0000000000005640], Graphs in FIGS. 4A and 4B show Kaplan-Meier curves for patients with phenylalanine level at baseline (Phase) greater than or equal to 11.2 pmol / dL versus Phase less than 11.2 pmol / dL (FIG. 4A) and for patients with maximal phenylalanine level during the stay in the ICU (Pmax) greater than or equal tol 1.2 pmol / dL versus Pmax less thanl 1.2 pmol / dL (FIG. 4B). The schematic in FIG.4C shows synthesis and recycling pathways of the tetrahydrobiopterin (BH4) and pteridine system. Graphs in FIGS. 4D and 4E show Kaplan-Meier curves for patients with Genetic Risk Score (GRS) greater than or equal to 2 versus GRS less than 2 in patients with Phase less than 11.2 pmol / dL (FIG. 4D) and in patients with Phase greater than or equal to 11.2 pmol / dL (FIG.4E). In FIGS. 4A-E, AKR = aldose reductase, BH2 = dihydrobiopterin, CBR = carbonyl reductase, DHFR = dihydrofolate reductase, DHPR = dihydropteridine reductase, GTP = guanosine triphosphate, GTPCH = GTP cyclohydrilase, PAH = phenylalanine hydroxylase, PCD = pterin-4a-carbinolamine dehydratase, PTPS = 6-pyruvoyl tetrahydropterin sunthase, qBH2 = quinonoid BH2, SR = sepiapterin reductase. F-OXPH4 = 6-(l’-oxo-2’-hydroxypropyl)-tetrahydropterin, and 2’-OXPH4 = 6-(l’-hydroxy-2’-oxopropyl)-tetrahydropterin. [See Wang, et al.]

[0039] Phe also has correlation with other disease states such as those where inflammation is implicated. Rheumatoid arthritis is a chronic autoimmune disease with complex causes and recurrent attacks that can easily develop into chronic arthritis and eventually lead to joint deformity. Inflammation causes dysregulation of Phe metabolism and elevated Phe levels. For example, see [Jian, C., Wei, L., Wu, T. et al. Comprehensive multi-omics analysis reveals the core role of glyccrophospholipid metabolism in rheumatoid arthritis development. Arthritis Res Ther 25, 246 (2023). https: / / doi.org / 10.1186 / sl3075-023-03208-2],

[0040] Kidney disease or function will also affect Phe levels in the body through altered metabolisms or clearance. This is due to an impairment in the conversion of phenylalanine to tyrosine, a process that is typically facilitated by the enzyme phenylalanine hydroxylase.Therefore, Phe or Tyr or their ratio may correlate with inflammation, cytokines, or C-reactiveprotein. In individuals with Chronic Kidney Disease (CKD), phenylalanine (Phe) levels tend to be normal or slightly elevated, while tyrosine (Tyr) levels are often decreased, leading to a reduced Tyr / Phe ratio (due to the impairment in the conversion of phenylalanine to tyrosine)Therefore, Phe or Tyr or their ratio may correlate with kidney function, creatinine, cystatin-C, or blood-urea-nitrogen (BUN). And so, Phe or Tyr may also represent or correlate to a least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine such as IL-6, C-reactive protein or TNF-a, kidney function, creatinine cystatin-C, blood-urea-nitrogen (BUN).

[0041] However, all of the data sets shown in the graphs of FIGS. 1 A-4E and other mentioned studies are based on single-data point Phe measures via a blood draw, and in many cases ratios of Phe to other amino acids are required to correct for confounding effects such as diet, kidney function, and other factors. Even ratios are subject to confounding factors such as diet and kidney clearance. As such, the use of Phe and / or other amino acids for possible correlation to health issues (such as cardiovascular issues) suffers the problems of being invasive, time-consuming, inaccurate due to multiple factors that can confound interpretation of amino-acid concentrations, and requiring scheduling and travel of the subject (such as to a clinic or medical facility for blood draw).

[0042] Aspects of the present invention however, overcome the drawbacks described above by providing methods, apparatus, and systems for monitoring the amount or concentration of at least one amino acid from a subject (e.g., in a sample fluid such as interstitial fluid), including continuous monitoring, which results in multiple data points being taken over a period of time. By these methods, apparatus, and systems, aspects of the present invention provide continuous or periodic measurements of the at least one amino acid (as opposed to the single data point measurements seen in current methods and apparatus) and provide same in a more readily accessed fluid (e.g., interstitial fluid) that does not require the same level of invasiveness, time consumption, etc. as currently seen with blood draws.

[0043] Thus, an amino acid monitoring system of the present invention provides distinct advantages in user ergonomics, cost, by providing trending data, by providing real time data, or other advantages as taught herein. Several additional advantages of the present invention include the following: First, the present invention may include a Circadian Fasting Advantage. For example, in one embodiment, the present invention is configured to prioritize or exclusively utilize biochemical data captured during a physiological fasting window, specifically during the subject’s nocturnal sleep cycle. By establishing a 'nightly fasting baseline,' the system eliminates transient metabolic noise associated with post-prandial (post-meal) spikes in amino acid concentrations, such as phenylalanine. This nightly synchronization allows the system to detectsubtle, endogenous shifts in metabolic pathways (e.g., the phenylalanine-to-tyrosine conversion rate) that are indicative of hepatic congestion and systemic cardiac stress, which would otherwise be masked by dietary intake during waking hours.

[0044] The present invention may also include the combination of at least one amino acid measure and at least one other non-amino acid measure, such as another molecule or a more conventional wearable non-molecular measure such as heart rate, blood pressure, or physical activity. The present invention may therefore include Multivariate Pattern Recognition. For example an amino acid (e.g., phenylalanine, Phe), a renal marker (e.g., creatinine, Cr), and an electrolyte (e.g., potassium, K+) can achieve high clinical specificity of AUROC >0.9 for heart failure using the probability P of an impending clinical event is determined by a weighted risk score Z, defined by the following logistic regression equation Z=Po+(5i(APhenightiy)+|32(ACrnightiy )+p3(AKmhtiy+) where A represents the 48-to-72-hour velocity of the analyte concentration relative to a personalized 14-day nocturnal (nightly) fasting baseline. The present invention may therefore include at least one additional measure monitored beyond an amino acid and performing multivariate pattern recognition on the measures of the amino acid and the at least one additional measure.

[0045] The present invention can utilize logical gating wherein a clinical alert is only triggered if a multivariate pattern is recognized, such as a simultaneous increase in the velocity of phenylalanine and creatinine concentrations coupled with a deviation in potassium levels. This triad reflects the 'Cardiorenal-Metabolic' axis, allowing the system to distinguish between a localized blip in a single analyte and a systemic decompensation event requiring medical intervention. The present invention may also include Triage Togic and Alert Tiering: a multitiered alert architecture designed to minimize clinician alert fatigue while maintaining a high area under the receiver operating characteristic curve (AUROC). For example, the present invention can provide an alert to the patient or to the doctor using a specificity- and time-weighted threshold such as AUROC >0.92, and maintaining the AUROC for > 2 days of data before notifying medical personnel. By optimizing the threshold specifically for high-risk, fragile populations, the system achieves an economic 'sweet spot' that minimizes the combined costs of false alarms and hospitalizations. The present invention may therefore comprise at least one time-weighted threshold and gating of at least one alert.

[0046] One particular aspect of the present invention is directed to a method for determining whether a subject is experiencing, has experienced, or is at risk of experiencing a health issue (e.g., cardiovascular issue). The method includes the steps of (1) monitoring the amount or concentration of at least one amino acid in a subject, wherein the at least one amino acid is an amino acid that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine (such as IL-6 or TNF-a), C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen (BUN); (2) detecting a deviation in the amount or concentration of the at least one amino acid away from a baseline value of amount or concentration of the at least one amino acid; (3) measuring a change in the amount or concentration of the at least one amino acid after detecting the deviation away from the baseline value of amount or concentration of the at least one amino acid; and (4) determining whether the subject is experiencing, has experienced, or is at risk of experiencing a changing (e.g., worsening or improving) condition or health status based on the measured change of the amount or concentration of the at least one amino acid.

[0047] Another aspect of the present invention is directed to a method of determining whether a subject is experiencing, has experienced, or is at risk of experiencing a health issue. The method includes the steps of (1) continuously monitoring at least one amino acid in a subject via a plurality of measurements of the amount or concentration of the at least one analyte in the subject, wherein the at least one amino acid is an amino acid that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine (such as IL-6 or TNF-a), C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen (BUN); (2) detecting a change in the amount or concentration of the at least one amino acid in the subject over the plurality of measurements, the change being an increase or a decrease in the amount or concentration of the at least one analyte; and (3) determining whether the subject is experiencing, has experienced, or is at risk of experiencing a changing (e.g., worsening or improving) condition or health status based on the change of the amount or concentration of the at least one amino acid.

[0048] Yet another aspect of the present invention is directed to a method of determining whether a subject is experiencing, has experienced, or is at risk of experiencing a health issue (such as a worsening condition or health status). The method includes the steps of (1) monitoring the amount or concentration of at least two amino acids in a subject, wherein the at least two amino acids have a ratio that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine, C-reactive protein, kidney function, creatinine cystatin-C, blood-urea-nitrogen; (2) detecting a deviation in the ratio away from a baseline ratio; and (3) measuring a change in the ratio after detecting the ratio; and (4) determining whether the subject is experiencing, has experienced, or is at risk of experiencing at least one of a changing (e.g., worsening or improving) condition or health status based on the measured change in the ratio.

[0049] As an example of how amino acids may be used relative to a health issue, as described above, one amino acid that has been shown to correlate to cardiovascular issues is phenylalanine(Phe). And so, in one embodiment of the present invention, the amino acid being monitored is Phe. In further embodiments, an additional or alternative amino acid that can be monitored is leucine (Leu) and / or tyrosine (Tyr) which is the metabolite of Phe. And, in further embodiments, more than one amino acid can be monitored (e.g., monitoring of Phe and Leu). And in still further embodiments, a ratio of at least two amino acids (e.g., Phe and Leu, Phe and Tyr, Leu and Tyr) can be monitored. And in certain embodiments, the health issue being monitored may be for a cardiovascular issue.

[0050] A device may be used to obtain sample from a subject (such as interstitial fluid), with the device then detecting the amount and / or concentration of at least one amino acid (e.g., Phe) in the sample. Various such devices may be used for monitoring the amino acid in the subject. Below are examples of embodiments and configurations that such devices (or systems including such devices) may take. And so, they are not intended to be limiting on the particular structure or type of device to be used (or components of the device or of a system including a device). In certain embodiments, the device may be an affinity based sensor. And in specific embodiments, the affinity based sensor may be an electrochemical aptamer sensor.

[0051] Certain non-limiting examples of electrochemical aptamer based sensors are as follows: With reference to FIG. 5, an example of a sensor device 100 as placed initially in a sample fluid such as dermal interstitial fluid of skin 12 is shown. The device includes a micro needle support 110 that can be made of metal, semiconductor, or plastic for example, and at least one working electrode 120 such as gold, carbon, platinum, or other suitable electrode material. Counter and references electrodes are not shown and may be included on feature 110. The device may also comprise electronics for reading the sensor 120 and communicating data to a user or smart phone (not shown). Sensor 120 may also be an aptamer sensor comprising at least one blocking layer a plurality of molecules such as mercaptohexanol or mercaptooctanol that are thiol bonded to the electrode, and at least one aptamer that is responsive to binding to an analyte and which contains a redox tag such as methylene blue. The working electrode(s) 120 are typically for the same analyte, such as Phe for example, and are embedded through the epidermis 12a and in the dermis 12b, and potentially into the hypodermis 12c. The depth of penetration into skin 12 by the device 100 is typically 100’s of pm (often 500-700 pm) for micronccdlc arrays as shown in FIG. 5. With further reference to FIG. 5, the device 100 may also use hollow microneedles and a sensor which is inside the hollow microneedles or which is outside the body (not shown), as taught for monitoring of Phe by, Friedel M, Werbovetz B, Drexelius A, Watkins Z, Bali A, Plaxco KW, Heikenfeld J. Continuous molecular monitoring of human dermal interstitial fluid with microneedle-enabled electrochemical aptamer sensors. Lab Chip. 2023 Jul 12;23(14):3289-3299. doi: 10.1039 / d31c00210a. PMID: 37395135.[0052| With reference to FIG. 6, where like numerals refer to like features, a conventional prior art sensor device 200 includes a single needle or microstrip element with a working electrode 220 embedded in the hypodermis 12c. This arrangement is commonly employed in continuous glucose monitors. The working electrode 220 is embedded deeply enough with adequate penetrating depth (e.g. ~5 mm) such that for most users of the device 200 the working electrode 220 will always be securely in the hypodermis 12c during use of the device 200.

[0053] With reference to FIG. 7, where like numerals refer to like features, for embodiments of the present invention additional details are provided on example working electrode construction and operation (with particular reference to a device that is an affinity -based sensor -such as an aptamer sensor). The working electrode 320 is comprised of an electrode material such as gold. The gold is then incubated with aptamers 350 via thiol attachment to the gold electrode, and the aptamer includes a redox tag such as methylene blue 352. The aptamers may have a sequence of CG ACC GCG TTT CCC AAG AAA GCA AGT ATT GGT TGG TCG [SEQ ID NO. 1], Alternative sequences include and are not limited to: CCG CGT TTC CCA AGA AAG CAA GTA TTG GTT GGT C [SEQ ID NO. 2], AGC GTT TCC CAA GAA AGC AAG TAT TGG TTT [SEQ ID NO. 3], CCG GTG GGG GTT CTT TTT CAG GGG AGG TAG GGT C [SEQ ID NO. 4], CGA CGA GGC TGG ATG CAT TCG CCG GAT GTT CGA TGT CG [SEQ ID NO. 5], and AGA GGC TGG ATG CAT TCG CCG GAT GTT CGA TT [SEQ ID NO. 6]. In between the aptamers the gold may be further incubated with a protective monolayer 356 such as mercaptohexanol, mercaptoocotanol, or other suitable chemistry. A protective membrane (not shown) such as UV-cured polybetaine hydrogel or other suitable material may be added to prevent fouling of the monolayer surface. The working electrode 320 may be preserved in a preservative such as trehalose to enable dry storage. In a non-limiting but specific example, binding of aptamer 350 to target 354 causes a shape confirmation change which alters the distance between the redox tag such as methylene blue 352 and the electrode 358 resulting in a change in electron transfer (a change in electrical current) - such as, for example, an increase in electrical cunent. The cunent can be measured using square wave voltammetry or other suitable techniques. As concentration of target 354 increases, more binding of target 354 to aptamer 350 occurs, and more electron transfer occurs (more measurable electrical current). As concentration of target 354 decreases, conversely electrical current decreases.

[0054] An alternate working electrode fomiat can be created [such as that described in A. Ferlazzo, C. Espro, D. lannazzo and G. Neri, "Determination of Phenylalanine by a Novel Enzymatic PHD / SPE Biosensor," in IEEE Transactions on Instrumentation and Measurement, vol. 72, pp. 1-8, 2023, Art no. 9508308, doi: 10.1109 / TIM.2023.3284027], An enzymatic Phe biosensor can include a working electrode functionalized with the enzyme Phe dehydrogenase(PHD) or another suitable enzyme for Phe. The enzyme will catalyze the transformation of Phe to pyruvic acid, NADH, and ammonia, and can be measured, for example with chronoamperometry or with open circuit potential (OCP) potentiometric techniques over a relatively wide concentration range (0- 5000pM). Devices as taught herein can insert the supports carrying the working electrodes into skin using one or methods such as those commonly deployed for the insertion of glucose sensors needles for continuous glucose monitors (such as a slotted insertion guide or other methods).

[0055] In fact, in another embodiment, glucose sensing may be paired with amino acid sensing on a single sensing device. For example, in FIG. 5, one of the electrodes 120 could be dedicated to Phe sensing and one to glucose using well known glucose enzymatic sensing chemistry. The advantage of Phe sensing for such an application is that many diabetics have heart disease as a comorbidity, and suffer from inflammation or infections that a rise in Phe concentrations can also correlate with. And so, by combining Phe and glucose sensing on a single device, diabetics could have a simple wearable device that not only senses glucose, but which also warns of worsening health with comorbidities such as heart failure, infection, or other changes in health status that would prompt bringing the patient into the clinic for further evaluation. This embodiment of amino acid sensing combined with glucose sensing is nonlimiting, such that amino acid sensing could be combined with sensing of other non-amino acid analytes in a single device.

[0056] In such examples of devices, electrochemical measurements can be performed with a miniaturized potentiostat or performed by a benchtop CHI 620E potentiostat (commercially available from CH Instruments, Inc., of Austin, Texas) connected to a 64-channel multiplexer in a standard three-electrode system with aptamer / alkylthiolate functionalized electrodes serving as working electrodes. The counter and reference electrodes can be inserted into the skin using a platinum counter electrode, and a Ag / AgCl reference electrode, or alternately the counter and reference electrodes can be a large gel-electrode -pad electrode on the surface of the skin as taught in PCT / US21 / 51972 -‘APTAMER SENSORS WITH REFERENCE AND COUNTER VOLTAGE CONTROL’. Cyclic voltammograms can be recorded in a window from -0.1 V to -0.5 V at a scan rate of 100 mV / s. Square- wave voltammetry can be performed in a potential window from -0.1 V to -0.5 V at 25 mV amplitude at the optimal frequency of measurement for each aptamer.

[0057] Devices used in the method of the present invention are not limited to specific examples as taught herein.

[0058] With a basic understanding of the above-described sensors in place, reference is now drawn to FIG. 8, which illustrates a general diagram of a computer system 800 according tovarious aspects of the present disclosure. Like numerals in FIG. 8 not necessarily refer to like features like that in the other figures. The computer system 800 comprises a plurality of hardware processing devices (designated generally by the reference 802, 804) that are linked together by one or more network(s).

[0059] The network(s) provides communications links between the various processing devices 802, 804 and may be supported by networking components 807 that interconnect the processing devices 802, 804, including for example, routers, hubs, firewalls, network interfaces, wired or wireless communications links and corresponding interconnections, cellular stations and corresponding cellular conversion technologies (e.g., to convert between cellular and TCP / IP, etc.). Moreover, the network(s) may comprise connections using one or more intranets, extranets, local area networks (LAN), wide area networks (WAN), wireless networks (Wi-Fi), the Internet, including the world wide web, cellular and / or other arrangements for enabling communication between the processing devices 802,804, in either real time or otherwise (e.g., via time shifting, batch processing, etc.). Data can be processed, stored, communicated or other functions in the cloud 806.

[0060] A processing device 802, 804 can be any device capable of communicating with another processing device 802, 804, e.g., via Bluetooth, Ultrawide band, near field communication (NFC), via one or more radio frequencies (RF) or via any other form of wired or wireless communication, over the network, or combinations thereof.

[0061] Some examples of processing devices 804 are cellular devices (including cellular mobile telephones (i.e., smartphones)), tablet computers, netbook computers, notebook computers, personal computers, servers, cloud devices, edge devices, etc.

[0062] Also, in certain contexts and roles, a processing device 802 is intended to be a wearable monitoring device. Examples of a wearable monitoring device include a purpose-driven appliance, Internet of Things (loT) device, special purpose device, etc. A processing device 802 implemented as a wearable monitoring device is schematically illustrated in FIG. 8 as a wearable device mounted to a patient’s arm solely for convenience of illustration. In practical applications, the wearable monitoring device can attach to other parts of a patient’s body.

[0063] In some embodiments, the wearable monitoring device can communicate locally (e.g., to a smart phone) via Bluetooth, ultrawidc band, via one or more radio frequencies (RF) or via any other form of wired or wireless communication. In other embodiments, the wearable monitoring device can communicate across a network, e.g., via Wi-Fi and / or communicate locally to another processing device 802.

[0064] The illustrative computer system 800 also includes a processing device implemented as a server 812 (e.g., a web server, file server, and / or other processing device) that supports ananalysis engine 814 and corresponding data sources (collectively identified as data sources 816). The analysis engine 814 and data sources 816 provide the resources to implement and store data related to collecting and aggregating data from wearable monitoring devices, captured events, combinations thereof, etc., as described in greater detail herein.

[0065] In an exemplary implementation, the data sources 816 are implemented by a collection of databases that store various types of information. Solely by way of example, the data sources 816 can include device data 818, e.g., data related to wearable monitoring devices, including configuration data, version data, software versioning and control, data generated from wearing a wearable monitoring device, etc. The data sources 816 can also include medical data 820, e.g., medical research, etc., used to calibrate, tune, design, modify, etc., wearable monitoring devices. The data sources 816 can also optionally include user data, e.g., data regarding the patients that are wearing the wearable monitoring devices, where such data is collected. As yet further examples, the data sources 816 can include platform data 824, e.g., data used by the analysis engine 814, e.g., computer drivers, GUI information, algorithms for processing physiological conditions, etc. As yet a further example, the data sources 816 can optionally include miscellaneous data 826, e.g., any data needed by the analysis engine 814 that is not otherwise accounted for above.

[0066] Considering FIG. 8 as an environment used by wearable monitoring devices, in some embodiments, the processing of physiological data of a corresponding patient wearing the wearable monitoring device (e.g., biochemical sensing with additional sensing modalities that enhance patient care or health and wellness) can be carried out entirely on a processing device 802 (such as a wearable monitoring device itself); on a processing device 802 such as a smartphone, by the analysis engine 814, or via combinations thereof (e.g., by distributing processing tasks among two or more processing devices).

[0067] With specific regard to a processing device 802 implemented as a wearable monitoring device (see processing device 802 schematically attached to a patient's arm), it may be desirable to carry out all of the processing on the wearable monitoring device itself. In this regard, a corresponding device such as a smartphone can optionally provide a graphical user interface for displaying dashboard measurement results, but all processing is carried out on the wearable monitoring device itself.

[0068] In other embodiments, the smart phone can carry out some processing, e.g., to compare computed data to dashboard thresholds, to carry out algorithms, rules, or other processing, as described more fully herein.

[0069] In still other embodiments, the analysis engine 814 can collect data from each wearable monitoring device, e.g., for trend analysis of patient data, for device state of healthmonitoring (e.g., to detect faults in the wearable devices themselves), for battery charge level monitoring, for versioning (such as to carry out software updates), etc.

[0070] In some embodiments, the analysis engine 814 is controlled by a third party, e.g., the manufacturer of the wearable monitoring devices that are implemented in the environment.

[0071] In some embodiments, the analysis engine 814 schematically represents integration into an electronic health record system, e.g., to connect a patient to the patient’s doctor so that the doctor can access the electronic data generated by a corresponding wearable monitoring device.

[0072] Referring now to FIG. 9, an example wearable monitoring device 900 is schematically illustrated, according to aspects of the present disclosure. Like numerals in FIG. 9 not necessarily refer to like features like that in the other figures. The wearable monitoring device 900 can represent an example embodiment of a processing device 802 (FIG. 8), e.g., a wearable monitoring device as previously described.

[0073] The wearable monitoring device 900 includes a housing 910 that attaches to a patient. The housing can attach to the patient via an adhesive 904, a strap, or other securement.

[0074] The wearable monitoring device 900 also includes at least a first working electrode 920 and may include a second working electrode 922 and further may include a third working electrode 923 or even more working electrodes. In some embodiments, one or more electrodes include an analyte detecting material, e.g., aptamers or enzymes or ion-selective electrodes, or other suitable materials such that continuous sensing can be carried out for at least one analyte such as an amino-acid and in some embodiments at least one additional analyte that is not an amino-acid. Electrode 950, as previously described may be a gel electrode pad and serve the roles of a reference and counter electrode.

[0075] In practical applications, the housing 910 is couplable to the electrodes 920, 922, 924. As used herein, "couplable" is to be constmed broadly to mean any one of permanently coupled, detachably coupled, temporarily coupled, user attachable, user detachable, user attachable and detachable, factory attachable, factory detachable, user attachable, factory attachable and detachable, or any combination thereof, unless specifically noted otherwise.

[0076] As illustrated, the housing 910 includes a potentiostat 991 that is communicably coupled to the electrodes 920, 922, 924 (or a combination thereof) using an optional multiplexer 990, or alternatively each of electrodes 920, 922, 924 can receive a direct dedicated connection to a potentiostat 991. In practical applications, the term “potentiostat” is to be interpreted broadly, and is not limited to any particular number of sensors. For instance, the potentiostat can be implemented as a bipotentiostat, polypotentiostat, etc., depending upon the sensor configuration provided by the wearable monitoring device 900.[00771 Additionally, wearable monitoring device 900 includes a controller 993 that is communicably coupled to memory 992. The controller 993 is also communicably coupled to a communication interface 994.

[0078] The controller 993 includes necessary electronics that enable the controller 993 to carry out the intended functionality of the wearable monitoring device. For instance, the controller 993 can include a processor, bus interface, ports, registers, memory, etc., that enables the wearable monitoring device 900 to carry out the functionality described more fully herein.

[0079] Also, as illustrated, the controller 992 is communicably coupled to one or more of the optional multiplexer 990, potentiostat 991, the memory 992, the transceiver(s) 994, optional miscellaneous sensors 995, optional display / output 996, combinations thereof, etc.

[0080] The communication interface 994 may comprise, for example, at least one transceiver that communicates via Bluetooth, Wi-Fi, Ultrawideband, near field communication, combinations thereof, etc.

[0081] The optional display / output 996 can comprise a display screen, a dimensionally limited display screen, a touch screen, a haptic output, a light output, a speaker / alarm, or combinations thereof.

[0082] The controller 993 uses the potentiostat 991 to collect measurements from electrodes 920a, 920b, 920c, and stores the collected measurements in the memory 992. The controller 993 may further provide filtering, analysis, control, authorization, authentication, and other controller specific functions. The communication interface 994 facilitates coupling the wearable monitoring device 900 with an external computing device, e.g., a smartphone, a cloud computer, etc. In this regard, the communication interface 994 can include one or more modalities, each with different data and / or authorizations. For instance, a patient may access data from the wearable monitoring device on a smartphone, whereas a doctor may be able to access more detailed information from a cloud server and / or through electronic health records (see FIG. 9). In this regard, multiple modalities of communication may be utilized with wearable monitoring device 900.

[0083] In some embodiments, the adhesive 904 of the wearable device 900 is, or includes, a gel electrode 950 that is connected to at least one of the potentiostat 991, the controller 993, or the sensor 995. For example, a gel electrode 950 could be the counter or reference electrode for the electrodes 920, 922, 924.

[0084] As noted above, the device that is used with the methods and systems described herein need not include an electrochemical aptamer sensor, as other types of devices are contemplated. For example, another type of device that may be used is an enzymatic biosensor, such as that described in Ferlazzo, et al.. Determination of Phenylalanine by a Novel Enzymatic PHD / SPEBiosensor, IEEE Transactions on Instrumentation and Measurement, vol. 72, pp. 1-8, 2023 (incorporated by reference herein). Yet another example of a type of device that may be used is a sensor such as that described in Huang, et al., Determination of L-Phenylalanine Based on an NADH-Detecting Biosensor, Anal. Chem., 1998, 70, 5, pp. 991-997 (incorporated by reference herein).

[0085] With reference to previous FIGS. 1-9, sensors measuring an amino acid or amino acid ratios can be utilized to measure or predict health or disease status. As has been described above, previous devices have not yet been successful in achieving an acceptable level of accuracy for reasons such as (1) monitoring accuracy may be limited by blood to interstitial fluid correlation, or by the electrochemical sensor itself, or other factors, and (2) data sets are based on single-data point amino acid (e.g., Phe) measures via a blood draw, and in many cases Phe to other amino acid ratios are required to correct for confounding effects such as diet, kidney function, and other factors.

[0086] In contrast, the various aspects of the present invention overcome the drawbacks of cunent uses of biomarkers for determination of health issues.

[0087] As described above, a first aspect of the present invention is directed to a method comprising (1) monitoring the amount or concentration of at least one amino acid in a subject, wherein the at least one amino acid is an amino acid that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine (such as IL-6 or TNF-a), C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen (BUN); (2) detecting a deviation in the amount or concentration of the at least one amino acid away from a baseline value of amount or concentration of the at least one amino acid; (3) measuring a change in the amount or concentration of the at least one amino acid after detecting the deviation away from the baseline value of amount or concentration of the at least one amino acid; and (4) determining whether the subject is experiencing, has experienced, or is at risk of experiencing a changing (e.g., worsening or improving) condition or heath status based on the measured change of the amount or concentration of the at least one amino acid. The worsening condition or health status, in certain embodiments, may include a cardiovascular issue. This aspect of the present invention includes a method which results in a comparison of amounts or concentrations of the at least one amino acid to some baseline value, and making a determination about the subject based on the comparison.

[0088] A second aspect of the present invention is directed to a method that does not necessarily require a baseline value or profile that a subject’s amount or concentration of amino acid is then compared to. Rather, in this second aspect, the determination regarding health issues (e.g., a cardiovascular issue) may come from a trend line of the data points being collected as theamount or concentration of the at least one amino acid is being measured. This aspect then is directed to a method comprising (1) continuously monitoring at least one amino acid in a subject via a plurality of measurements of the amount or concentration of the at least one analyte in the subject, wherein the at least one amino acid is an amino acid that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine (such as IL-6 or TNF-a), C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen (BUN); (2) detecting a change in the amount or concentration of the at least one amino acid in the subject over the plurality of measurements, the change being an increase or a decrease in the amount or concentration of the at least one analyte; and (3) determining whether the subject is experiencing, has experienced, or is at risk of experiencing a changing (e.g., worsening or improving) condition or health status (e.g., a cardiovascular issue) based on the change of the amount or concentration of the at least one amino acid.

[0089] Yet another aspect of the present invention is directed to a method of determining whether a subject is experiencing, has experienced, or is at risk of experiencing a health issue (such as a worsening condition or health status). The method includes the steps of (1) monitoring the amount or concentration of at least two amino acids in a subject, wherein the at least two amino acids have a ratio that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine, C-reactive protein, kidney function, creatinine cystatin-C, blood-urea-nitrogen; (2) detecting a deviation in the ratio away from a baseline ratio; and (3) measuring a change in the ratio after detecting the ratio; and (4) determining whether the subject is experiencing, has experienced, or is at risk of experiencing at least one of a changing (e.g., worsening or improving) condition or health status based on the measured change in the ratio.

[0090] Turning to the first aspect of the invention, determining whether the subject is experiencing, has experienced, or is at risk of experiencing a health issue may further include determining a magnitude of response. This magnitude of response, in one embodiment, can be calculated as the change in the amount or concentration of the at least one amino acid between the baseline value and a peak or trough amount or concentration. It will be recognized by those of ordinary skill in the art that the measured change of the amount or concentration of the at least one amino acid in the subject can be an increase in the amount or concentration of the at least one amino acid in the subject, or can be a decrease in the amount or concentration of the at least one amino acid in the subject. Determination of experience or risk of experience of a cardiovascular issue may turn on whether there is an increase or decrease, or on the magnitude of the increase or decrease, or rate of increase or decrease (or combinations of these factors).[00911 The measured change of the amount or concentration of the at least one amino acid in the subject may be measured and / or reported in different ways as well. For example, in one embodiment, the change is measured as a ratio of the measured amount or concentration of the at least one amino acid to the baseline value of amount or concentration of the at least one amino acid. In one specific example of this embodiment, the at least one amino acid may be phenylalanine, and the ratio (for the measured change in amount or concentration of Phe) is greater than or equal to 0.6 as compared to baseline value (when measured for a cardiovascular issue).

[0092] In other embodiments, the change may be measured and / or reported as a numerical value. In one specific example of this embodiment, the at least one amino acid may be phenylalanine, and the measured amount or concentration of phenylalanine is a measured concentration greater than or equal to 65.6u M (when measured for a cardiovascular issue).

[0093] Considering the above then, for example, a Phe sensing system could provide a daily report of Phe from moving averaging, minimum Phe measurement, or other types of data interpretations or measurements that would reveal a Phe level of >65.6 pM and therefore a 0.6 rating for accumulative survival as illustrated in FIGS. 1A-C. This 0.6 ratio is of baseline Phe concentration to increased Phe concentration when measured (which could also be expressed as a ratio of measured Phe to baseline, which would be 1 / 0.6 or approximately 1.67). Thus, in various embodiments the ratio used for determination of an issue (such as experienced or risk of a cardiovascular issue) can be of measured Phe to baseline Phe, and can be greater than or equal to 1.2, 1.3, 1.5, 1.6, 1.67, or 2.0.

[0094] Further, for example, there is a pathogenic role for increased phenylalanine levels in cardiac aging, linking plasma phenylalanine levels to cardiac senescence via dysregulated phenylalanine catabolism along a hepatic-cardiac axis. Thus, therapeutic strategies targeting dysregulated phenylalanine catabolism could be used in treating age-related / exacerbated cardiac dysfunction.

[0095] Next, as noted above, determining a cardiovascular issue by the methods of the first aspect of the present invention includes a comparison of a measured change in amino acid amount or concentration to a baseline value that allows one to determine a cardiovascular issue (whether current or past), or risk of same, based on magnitude of change from the baseline value, or rate of change from baseline value, or time a change is sustained over the baseline value, or rate of return to or near baseline value, or some combination of one, or more, or all of these factors.

[0096] In various embodiments of the first aspect of the present invention, then, there are multiple ways that the baseline value can be calculated, determined, or otherwise identified.Each of these methods generally fall into one of two overarching categories of baseline values: (1) those that are calculated based on the subject being monitored himself or herself, or (2) baselines that are generated from data obtained from an individual or populations of individuals external to the particular individual being monitored.

[0097] In the first category (baseline values generated based on data from the individual being monitored) one example would be a baseline that is established by a zero slope line extending from a data point of amino acid amount or concentration (e.g., phenylalanine) just prior to the increase of amino acid amount or concentration.

[0098] Another method for providing a baseline based on data generated by the individual themselves, would include the generation of a baseline profile individual to the subject being monitored.

[0099] And so, a baseline profile for the subject being monitored may be created via use of the device being used to calculate the cardiovascular issue via the methods of the present invention, or via use of another device. This can be done by monitoring the levels of the amino acid of interest for a period of time sufficient to create a useful profile. For example, the individual may be monitored for a period of one day, multiple days, one week, multiple weeks, one month, or multiple months in order to create a baseline profile of the amino acid in the individual. Such monitoring to create a profile may be performed using a device worn by the individual that periodically records the amount or concentration of amino acid in the individual (this device may also be the device that ultimately monitors the at least one amino acid in the subject following creation of a baseline profile). As mentioned, this may be recorded over a period of time sufficient to develop a baseline amino acid profile for the individual.

[0100] As noted above, in another category, the baseline profile may be created from an individual or population of individuals that do not include the subject being monitored. For example, in some embodiments, the baseline profile may be estimated based on relevant information about the individual, such as the person's age, weight, sleep habits, physical fitness, stress level, medications, etc. The individual's information may then be compared to aggregated data about other individuals in a database, and a baseline profile from a comparable individual or population of individuals would be selected. Alternatively (or additionally) an external baseline profile may selected from an individual or population of individuals who share at least one characteristic with the subject (e.g., from biological characteristics, like shared race, sex, BMI, etc.).

[0101] Once a profile is created or selected, that would be used as the baseline profile for comparing any monitored amount or concentration of amino acid. As amino acid (such as phenylalanine) fluctuates over the course of the day, one would monitor amounts orconcentrations of the amino acid compared to the amounts or concentrations shown on the profile at the same time of day as the monitoring occurs (e.g., if a reading of amino acid is taken at 10:00am, that result would be compared to the amount or concentration shown on the baseline profile at 10:00am).

[0102] In one embodiment, the baseline profile is the average amount or concentration of the at least one amino acid over a period of time. This average amount or concentration of the at least one amino acid over a period of time may be calculated from measured amounts or concentrations of the at least one amino acid in the subject over the period of time.Alternatively, the average amount or concentration of the at least one amino acid over a period of time may be calculated from measured amounts or concentrations of the at least one amino acid in a population of individuals over the period of time. In various embodiments, the period of time may be chosen from 1 hour, 4 hours, 12 hours, 24 hours, 2 days, 1 week, 2 weeks, and more than 2 weeks (though these time periods should be considered non-limiting, as other time periods may be used).

[0103] In another embodiment, the baseline profile is a plot of measured amounts or concentrations of the at least one amino acid per time of day of the at least one amino acid in the subject. In this embodiment, the baseline profile can be created by plotting measured amounts or concentrations of the at least one amino acid in the subject per time of day over a period of time. Alternatively, in this embodiment, the baseline profile may be created by plotting measured amounts or concentrations of the at least one amino acid in a population of individuals per time of day over a period of time. In either instance, creating the baseline profile may include measuring the amount or concentration of the at least one amino acid at least one time during each day for a plurality of days.

[0104] Further, the particular time during each day (whether one time or multiple times) for measurement of the amount or concentration of the at least one amino acid can be selected to provide a baseline profile that is not affected (or has a reduced effect) by or from physiological events that may cause a change in Phe levels, but which does not carry significance as to a cardiovascular event. So, for example, the particular time for measurement during the day (or during each day) may be during or near the end of nightly sleep, or can be prior to a meal or between meals. One may reduce confounding factors such as diet on Phe levels by automatically collecting Phe levels during periods in between meals, such as during or near the end of nightly sleep which mimics a ‘fasting Phe measurement’ without the need for the subject to fast. Such measurement of the amount or concentration of the at least one amino acid at these times may be initiated by the subject or, alternatively, may be initiated at a scheduled time by a device (i.e., anautomatic measurement). The period of time over which measurements can be taken can be 1 day, 2 days, 1 week, 1 month, or more than one month (though other periods times may be used).

[0105] In certain embodiments then, the baseline value to be used in calculating change of amount or concentration, is the amount or concentration of the at least one amino acid in the plot at the same time of day as the detected change in the amount or concentration of the at least one amino acid in the subject. In an embodiment, then, where the baseline profile has been created by measurements taken at time points, such as those above (during or near end of sleep; prior to or between meals), i.e., a time for maximum confidence of accuracy, then when monitoring the at least one analyte (e.g., Phe) a daily Phe measurement can be taken at one or more of the same times of maximum confidence for accuracy (e.g. near the end of sleeping) or at a minimum level during the day (such as in between meals) that provides a correlation with physiological status such as a status of heart failure.

[0106] Turning now to the second aspect: This aspect then is directed to a method comprising (1) continuously monitoring at least one amino acid in a subject via a plurality of measurements of the amount or concentration of the at least one analyte in the subject, wherein the at least one amino acid is an amino acid that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine such as IL-6, C-reactive protein or TNF-a, kidney function, creatinine cystatin-C, blood-urea-nitrogen (BUN); (2) detecting a change in the amount or concentration of the at least one amino acid in the subject over the plurality of measurements, the change being an increase or a decrease in the amount or concentration of the at least one analyte; and (3) determining whether the subject is experiencing, has experienced, or is at risk of experiencing a worsening condition or health status (e.g., a cardiovascular issue) based on the change of the amount or concentration of the at least one amino acid.

[0107] It will be recognized by those of ordinary skill in the art that the measured change of the amount or concentration of the at least one amino acid in the subject can be an increase in the amount or concentration of the at least one amino acid in the subject, or can be a decrease in the amount or concentration of the at least one amino acid in the subject. Determination of experience or risk of experience of a cardiovascular issue may turn on whether there is an increase or decrease, or on the magnitude of the increase or decrease, or rate of increase or decrease (or combinations of these factors).

[0108] Thus, for example, a method such as that of this second aspect of the invention can reduce confounding factors such as basal Phe levels and variance across the population by continuously or repeatedly measuring Phe to instead provide trending information which is better tailored to the invidual. For example, a patient may have a high normal level of Phe, but thatpatient may actually be recovering from heart failure which would be confirmed more strongly not by the actual concentration of Phe that is measured but by the fact that Phe is trending downwards over time.

[0109] The present invention further recognizes that phenylalanine (Phe) serves as a sensitive metabolic proxy for a plurality of systemic pathological states beyond isolated cardiovascular stress. Specifically, elevations in phenylalanine concentrations, particularly when measured during a nocturnal fasting window, act as an early-stage biomarker for systemic inflammatory response syndrome (SIRS) and sepsis. During high-state inflammatory events, pro-inflammatory cytokines (including IL-6 and TNF-a) impair the activity of the enzyme phenylalanine hydroxylase (PAH) in the liver, leading to a rapid accumulation of Phe in the blood and interstitial fluid. By monitoring the velocity of this accumulation, the system can differentiate between a chronic heart failure baseline and an acute septic event. Furthermore, because Phe metabolism is localized primarily in the liver, the system serves as a real-time monitor for congestive hepatopathy (liver congestion) and hepatic metabolic reserve. In subjects with co-morbidities such as Non-Alcoholic Steatohepatitis (NASH) or cirrhosis, the system utilizes the Phe-to-Tyr ratio as a proxy for hepatic functional capacity, providing a non-invasive 'metabolic stress test’ that tracks organ crosstalk between the heart, liver, and kidneys."

[0110] A Phe monitoring system can be configured to monitor the clinical activity and flarefrequency of autoimmune disorders, specifically Rheumatoid Arthritis (RA), by correlating nocturnal Phe kinetics with the Disease Activity Score 28 (DAS28). In RA patient populations, fasting Phe concentrations demonstrate a statistically significant positive con-elation with DAS28 scores (Spearman’s rho ~ 0.45-0.62, ) and serum C-Reactive Protein (CRP) levels. Specifically, a monitoring system can be calibrated to detect 'Active Disease' thresholds where Phe concentrations exceed a personalized baseline by at least 1 -20 pmol / L, or where the Phe / Tyrosine ratio increases by a factor of 1.4x or greater, indicative of IFN-y-induced BH4 cofactor depletion. By utilizing nightly longitudinal data, the system achieves an AUROC of 0.84 to 0.88 for distinguishing between 'Clinical Remission' (DAS28 < 2.6) and 'Moderate-to-High Disease Activity' (DAS28 > 3.2). This allows for the remote detection of inflammatory flares with a sensitivity of 82% and a specificity of 78%, providing a metabolic lead-time for therapeutic adjustment prior to the onset of debilitating joint swelling.

[0111] The system described herein is configured to achieve a diagnostic and prognostic accuracy, as measured by the Area Under the Receiver Operating Characteristic curve (AUROC), that significantly exceeds standard single-analyte snapshots. While a single -point measurement of phenylalanine (Phe) can provide a baseline AUROC of approximately 0.80 to 0.82 for heart failure diagnosis, the present invention’s integration of a multi-analyte'Cardiorenal-Metabolic Triad' — comprising Phe, creatinine, and potassium — specifically targets an AUROC in the range of 0.87 to 0.96. The present invention may therefore include determining a worsening condition that triggers an alert with an AUROC of at least 0.90 for at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine such as IL-6, C-reactive protein or TNF-a, kidney function, creatinine cystatin-C, blood-urea-nitrogen. By utilizing the temporal correlation between these analytes, particularly the simultaneous nocturnal fasting slopes of Phe and creatinine, the system identifies systemic decompensation with a specificity exceeding 90% while maintaining a sensitivity of at least 85%. This quantitative performance allows the system to function as a high-fidelity 'Digital NT-proBNP' equivalent, providing actionable clinical alerts with a lead time of 14 to 21 days prior to an acute event, thereby optimizing the healthcare economics of remote monitoring by minimizing the combined costs of false alarms and missed hospitalizations.

[0112] Embodiments of the present invention may further comprise at least one method for Signal Integrity and Error Mitigation. Example methods may include the following non-limiting examples of Impedance-Based Integrity Checks and Biological Velocity Limits. In Impedance-Based Integrity Checks, the sensor interface continuously monitors electrochemical impedance. Any sudden spike in impedance — often indicative of a "dry" sensor, physical dislodgement, or air bubble interference — triggers an automatic data-suppression flag rather than a clinical alert. In Biological Velocity Limits, the system applies "Physiological Plausibility Filters" based on the maximum known biological rates of change for phenylalanine, creatinine, and potassium. For example, a rise in creatinine of within a 1-hour window is statistically treated as a sensor drift or calibration error rather than a renal event, as it exceeds the possible rate of human organ failure.

[0113] Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventive elements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art.

Claims

WHAT IS CLAIMED IS:

1. A method comprising:monitoring the amount or concentration of at least one amino acid in a subject, wherein the at least one amino acid is an amino acid that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine, C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen;detecting a deviation in the amount or concentration of the at least one amino acid away from a baseline value of amount or concentration of the at least one amino acid;measuring a change in the amount or concentration of the at least one amino acid after detecting the deviation away from the baseline value of amount or concentration of the at least one amino acid; anddetermining whether the subject is experiencing, has experienced, or is at risk of experiencing at least one of a changing condition or health status based on the measured change of the amount or concentration of the at least one amino acid.

2. The method of claim 1, wherein determining whether the subject is experiencing, has experienced, or is at risk of experiencing a cardiovascular issue further comprises determining a magnitude of response, wherein the magnitude of response is the change in the amount or concentration of the at least one amino acid between the baseline value and a peak or trough amount or concentration.

3. The method of claim 1, wherein the measured change of the amount or concentration of the at least one amino acid in the subject is an increase in the amount or concentration of the at least one amino acid in the subject.

4. The method of claim 1 , wherein the measured change of the amount or concentration of the at least one amino acid in the subject is a decrease in the amount or concentration of the at least one amino acid in the subject.

5. The method of claim 1, wherein the measured change of the amount or concentration of the at least one amino acid in the subject is measured as a ratio of the measured amount or concentration of the at least one amino acid to the baseline value of amount or concentration of the at least one amino acid.

6. I’he method of claim 5, wherein the at least one amino acid is phenylalanine, and the ratio is greater than or equal to 1.2, 1.3, 1.5, 1.6, 1.67, or 2.0.

7. The method of claim 1, wherein the measured change of the amount or concentration of the at least one amino acid in the subject is measured as a numerical value.

8. The method of claim 1, wherein the at least one amino acid is phenylalanine, and the measured amount or concentration of phenylalanine is a measured concentration greater than or equal to 65.6pM.

9. The method of claim 1 , wherein the baseline value is the measured amount or concentration of the at least one amino acid that occurs just prior to a change in amount or concentration of the at least one amino acid.

10. The method of claim 1, wherein the baseline value is obtained from a baseline profile of amount or concentration of the at least one amino acid.

11. The method of claim 1, wherein the baseline profile is the average amount or concentration of the at least one amino acid over a period of time.

12. The method of claim 11, wherein the average amount or concentration of the at least one amino acid over a period of time is calculated from measured amounts or concentrations of the al least one amino acid in the subject over the period of time.

13. The method of claim 11, wherein the average amount or concentration of the at least one amino acid over a period of time is calculated from measured amounts or concentrations of the at least one amino acid in a population of individuals over the period of time.

14. The method of claim 11, wherein the period of time is chosen from 1 hour, 4 hours, 12 hours, 24 hours, 2 days, 1 week, 2 weeks, and more than 2 weeks.

15. The method of claim 10, wherein the baseline profile is a plot of measured amounts or concentrations of the at least one amino acid per time of day of the at least one amino acid in the subject.

16. The method of claim 15, wherein the baseline profile is created by plotting measured amounts or concentrations of the at least one amino acid in the subject per time of day over a period of time.

17. The method of claim 15, wherein the baseline profile is created by plotting measured amounts or concentrations of the at least one amino acid in a population of individuals per time of day over a period of time.

18. The method of claim 16 or claim 17, wherein creating the baseline profile comprises measuring the amount or concentration of the at least one amino acid at least one time during each day for a plurality of days.

19. The method of claim 18, wherein the time during each day for measurement of the amount or concentration of the at least one amino acid is during or near the end of nightly sleep.

20. The method of claim 18, wherein the time during each day for measurement of the amount or concentration of the at least one amino acid is prior to a meal.

21. The method of claim 18, wherein the measurement of the amount or concentration of the at least one amino acid is initiated by the subject.

22. The method of claim 18, wherein the measurement of the amount or concentration of the at least one amino acid is initiated at a scheduled time by a device.

23. The method of claim 18, wherein measuring the amount or concentration of the at least one amino acid at least one time during each day is at a plurality of times during each day.

24. The method of claim 23, wherein the plurality of times during each day includes a measurement during or near the end of nightly sleep and a measurement prior to a meal.

25. The method of claim 16, wherein the period of time is 1 day, 2 days, 1 week, 1 month, or more than one month.

26. The method of claim 15, wherein the baseline value is the amount or concentration of the at least one amino acid in the plot at the same time of day as the detected change in the amount or concentration of the at least one amino acid in the subject.

27. The method of claim 1, wherein the at least one amino acid is chosen from phenylalanine, leucine, and tyrosine.

28. The method of claim 1, wherein the at least one amino acid comprises two or more amino acids.

29. The method of claim 28, wherein the two or more amino acids includes two or more of phenylalanine, leucine, and tyrosine.

30. The method of claim 1, further comprising monitoring at least one non- amino acid analyte in the subject.

31. The method of claim 30, wherein the at least one non- amino acid analyte is at least one of glucose, creatinine, or potassium.

32. The method of claim 31, wherein the at least one amino acid is phenylalanine.

33. The method of claim 1, wherein the monitoring the amount or concentration of at least one amino acid in a subject is performed by a device including a sensor having at least one electrode.

34. The method of claim 33, wherein the sensor is an affinity-based sensor.

35. The method of claim 34, wherein the device includes a plurality of aptamers having affinity for the at least one amino acid.

36. The method of claim 35, wherein each aptamer of the plurality of aptamers is bound, directly or indirectly, to a surface of the at least one electrode.

37. The method of claim 35, further comprising a plurality of redox tags, wherein a redox tag of the plurality of redox tags is associated with each aptamer of the plurality of aptamers.

38. The method of claim 1, wherein the monitoring the amount or concentration of at least one amino acid in a subject is performed by an enzymatic sensor.

39. The method of claim 1, wherein the monitoring the amount or concentration of at least one amino acid in a subject is performed by a device including an NADH-detecting sensor.

40. The method of claim 1, further comprising monitoring at least one non-molecular biomarker in the subject.

41. The method of claim 1, further comprising at least one additional measure monitored beyond an amino acid and performing multivariate pattern recognition on the measures of the amino acid and the at least one additional measure.

42. The method of claim 1, further comprising at least one time- weighted threshold and gating of at least one alert.

43. The method of claim 1, wherein the determined worsening condition triggers an alert with an AUROC of at least 0.90 for at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine, C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen.

44. The method of claim 1, wherein the cytokine is IL-6 or TNF-a.

45. The method of claim 1, wherein determining whether the subject is experiencing, has experienced, or is at risk of experiencing a cardiovascular issue further comprises determining a magnitude of response, wherein the magnitude of response is the rate of return of the amount or concentration of the at least one amino acid to the baseline value from a peak or trough amount or concentration.

46. A method comprising:continuously monitoring at least one amino acid in a subject via a plurality of measurements of the amount or concentration of the at least one analyte in the subject, wherein the at least one amino acid is an amino acid that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine , C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen;detecting a change in the amount or concentration of the at least one amino acid in the subject over the plurality of measurements, the change being an increase or a decrease in the amount or concentration of the at least one analyte; anddetermining whether the subject is experiencing, has experienced, or is at risk of experiencing at least one of a changing condition or health status based on the change of the amount or concentration of the at least one amino acid.

47. The method of claim 46, further comprising a determination that the subject is experiencing, has experienced, or is at risk of experiencing a cardiovascular issue based on an increase of the amount or concentration of the at least one amino acid.

48. The method of claim 46, further comprising a determination that the subject is not experiencing, has not experienced, or is not at risk of experiencing a cardiovascular issue based on a decrease of the amount or concentration of the at least one amino acid.

49. The method of claim 46, wherein the plurality of measurements are taken over a period of time, and wherein the period of time is chosen from at least one hour, at least 12 hours, at least 1 day, at least one week, and at least one month.

50. The method of claim 46, wherein the at least one amino acid is chosen from phenylalanine, leucine, and tyrosine.

51. The method of claim 46, wherein the at least one amino acid comprises two or more amino acids.

52. The method of claim 51, wherein the two or more amino acids includes two or more of phenylalanine, leucine, and tyrosine.

53. The method of claim 46, further comprising monitoring at least one non-amino acid analyte in the subject.

54. The method of claim 53, wherein the at least one non-amino acid analyte isat least one of glucose, creatinine, or potassium.

55. The method of claim 53, wherein the at least one amino acid is phenylalanine.

56. The method of claim 46, wherein the monitoring the amount or concentration of at least one amino acid in a subject is performed by a device including a sensor having at least one electrode.

57. The method of claim 56, wherein the sensor is an affinity-based sensor.

58. The method of claim 57, wherein the device includes a plurality of aptamers having affinity for the at least one amino acid.

59. The method of claim 58, wherein each aptamer of the plurality of aptamers is bound, directly or indirectly, to a surface of the at least one electrode.

60. The method of claim 58, further comprising a plurality of redox tags, wherein a redox tag of the plurality of redox tags is associated with each aptamer of the plurality of aptamers.

61. The method of claim 46, wherein the monitoring the amount or concentration of at least one amino acid in a subject is performed by an enzymatic sensor.

62. The method of claim 46, wherein the monitoring the amount or concentration of at least one amino acid in a subject is performed by a device including an NADH-detecting sensor.

63. The method of claim 46, further comprising monitoring at least one non-molecular biomarker in the subject.

64. The method of claim 46, further comprising at least one additional measure monitored beyond an amino acid and performing multivariate pattern recognition on the measures of the amino acid and the at least one additional measure.

65. The method of claim 46, further comprising at least one time- weighted threshold and gating of at least one alert.

66. The method of claim 46, wherein the determined worsening condition triggers an alert with an AUROC of at least 0.90 for at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine , C-reactive protein, kidney function, creatinine cystatin-C, or blood-urea-nitrogen.

67. A method comprising:monitoring the amount or concentration of at least two amino acids in a subject, wherein the at least two amino acids have a ratio that correlates to at least one of cardiovascular function, NT-proBNP, BNP, cMyBP-C, MR-proANP, inflammation, a cytokine, C-reactive protein, kidney function, creatinine cystatin-C, blood-urea-nitrogen;detecting a deviation in the ratio away from a baseline ratio;measuring a change in the ratio after detecting the ratio; anddetermining whether the subject is experiencing, has experienced, or is at risk of experiencing at least one of a changing condition or health status based on the measured change in the ratio.

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