Non-invasive methods and devices for monitoring microbiome health

Abstract

The present disclosure is related to ex vivo methods and devices for identifying a presence of or predicting a risk for a condition in a subject, wherein the risk or condition is correlated with an oxidation-redox potential (ORP) of a biological sample obtained from the subject, such as a fecal sample. The methods and devices of the present disclosure can be used to diagnose and monitor gut microbiome health and assess the risk and / or presence of conditions such as obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes, hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof based on an ORP of the subject's sample.

Description

[0001] Attorney Docket No. 225003-701601

[0002] NON-INVASIVE METHODS AND DEVICES FOR MONITORING MICROBIOME HEALTH

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004]

[0001] The present disclosure claims priority to U.S. Application No. 63 / 716,028, filed on 4 November 2024, the contents of which is incorporated herein by reference in its entirety.

[0005] BACKGROUND

[0006]

[0002] An adult human gut is an extremely diverse ecosystem, harboring at least as many bacterial cells as the adult’s total number of somatic and germ cells. The collective genomes (referred to as the microbiome) contains over 1000-fold genes compared to the human genome.

[0007]

[0003] Studies have been conducted to show a link between gut microbiota, health, and disease, indicating that a host organism’s gut microbiota is an important modulator of its physiology and metabolism. The gut microenvironment is highly anaerobic, with redox potentials below -300 mV. These conditions favor growth of extremely oxygen-sensitive microbes and a healthy gut microbiota. However, oxygen may diffuse into the lumen from gut mucosa, generating an oxygen gradient from the gut mucosa to the lumen. The gut microbes achieve a reducing environment to protect themselves, and potentially the host, from oxidative stress.

[0008]

[0004] Comparative metagenomics has revealed that the microbiome of patients with type 2 diabetes, hyperlipidemia, inflammatory bowel disease and several other disease conditions are frequently associated with decreased microbial diversity and a reduced abundance of butyrate- producing bacteria, such as Faecalibacterium prausnitzii. There is thus a strong interest in exploring interactions between microbes as well as between microbes and hosts to deepen our understanding of health and disease conditions. Metagenomics is a powerful technology for studying the human gut microbiome composition and to predict its function. However, there are still some limitations in the use of metagenomics, for example, time constraints and costs. Furthermore, determining absolute quantification and comparing data across studies with different DNA extraction methods is challenging. Retrieving microbes as a pure culture is also challenging as a significant proportion of gut microbes are strict anaerobes.

[0009]

[0005] There thus remains a need for direct, quantitative, and user-friendly approaches in this field. Attorney Docket No. 225003-701601

[0010] SUMMARY

[0011]

[0006] To provide better insight into gut microbiota dynamics, Applicants developed a non- invasive validated, redox potential measuring device, (referred to herein in some embodiments as “ROXmeter”). This device measures a redox potential of a subject’s fecal sample as a proxy for information on the subject’s gut environment, microbial activity, and composition. Without wishing to be bound by theory, data generated from the device and methods of the present disclosure can be used to predict an onset, a presence, or a severity of one or more disorders, such as metabolic disorders, and help improve intervention therapies and subsequent outcomes by providing an understanding of gut microbiota dynamics.

[0012]

[0007] The present disclosure provides for study of the gut microbiome and diagnosis of diseases such as metabolic diseases using non-ingestible, ex vivo methods, systems, and devices that are suitable for all age groups. The technology of the present disclosure provides for easy-to-operate technology that is bio-fouling resistant and less influenced by spatial host derived redox active metabolites.

[0013]

[0008] The present disclosure relates to ex vivo methods for identifying a presence or a risk of a condition in a subject, wherein the risk or condition is correlated with an oxidation-redox potential (ORP) of a biological sample obtained from the subject.

[0014]

[0009] Moreover, the present disclosure describes systems and methods using the non-invasive ROXmeter to provide quick results that enable a real-time or near real-time clinical data for decision-making and on the need for therapeutic intervention. Continuous monitoring following the initiation of a therapy can then be performed at the bedside in a clinic, or even at home, without the need for complex equipment, or any reliance on centralized laboratory equipment. The data generated by the ROXmeter can be paired with smartphones or cloud platforms, supporting remote monitoring by medical personal, enabling early detection, a reduction in lab burden, and an overall cost saving in healthcare.

[0015]

[0010] Systems and methods comprising the non-invasive validated, redox potential measuring device, (ROXmeter) as described herein can be used for determining a need for treating and / or maintaining remission of a gastrointestinal disorder selected from inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory bowel disorder, irritable bowel syndrome, irritable bowel syndrome-diarrhea, irritable bowel syndrome-constipation, irritable bowel syndrome-mixed, irritable bowel syndrome-alternating, dyspepsia, gastro-esophageal reflux, diverticulitis, diverticular disease, gastroparesis, microscopic colitis, lymphocytic colitis, collagenous colitis, indeterminant colitis, eosinophilic esophagitis, HIV-associated diarrhea, antibiotic-associated colitis, Clostridium difficile-associated diarrhea, pseudo-membranous colitis, Atorney Docket No. 225003-701601 diarrhea associated with immunodeficiency disorders, small bowel overgrowth syndrome, celiac disease, Whipple's disease, CMV-associated colitis, Behcet's syndrome, and combinations thereof. [OH] The human gastrointestinal tract harbors approximately 1014bacteria that are composed of between 300 and 1,000 different species. A reduction in the normal levels and function of flora that occur naturally in the gastrointestinal tract of animals and in humans can cause various symptoms. Dysbiosis, defined as dysregulation of the normal homeostasis of the intestinal microbiota, has been implicated in the pathogenesis of myriad disease conditions, including, but not limited to, antibiotic-associated diarrhea (AAD), Clostridium difficile-associated disease (CDAD), acquired immunodeficiency syndrome (AIDS), hypothyroidism, food poisoning, obesity, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and colorectal carcinoma. Epidemiological studies have also linked altered composition of the intestinal microbiota with the development of rheumatoid arthritis, eczema, and other allergic diseases and aberrant microbiota during childhood may predispose one to the development of inflammatory gut diseases and diarrhea.

[0016]

[0012] Dysbiosis is a common condition that can have significant effects on health. However, diagnosing dysbiosis remains a challenge as there is currently no single definitive test. Dysbiosis may be treated by a variety of methods, including probiotics, which comprise administering live beneficial bacteria that can help restore the balance of the gut microbiota; prebiotics, which include non-digestible food components that promote the growth of beneficial bacteria; dietary changes, stress management; and antibiotics. The present disclosure provides simple point-of-care systems and methods for determining the presence of dysbiosis as well for monitoring a treatment used to treat the dysbiosis to determine whether a particular therapy should be stopped, maintained, or changed to a different treatment.

[0017]

[0013] For instance, in cases of severe dysbiosis caused by bacterial infections, antibiotics may be prescribed to target specific harmful bacteria. However, such antibiotics should be used with care and should be continuously monitored and limited as overuse of such a therapy could cause further disrupting the gut microbiota. The systems and methods of the present disclosure provide a means to not only determine whether a particular treatment is needed, but also once the treatment is started, the present systems and methods provide a means for monitoring the efficacy of the therapy and determining when to stop to avoid further disruption of the gut microbiota.

[0018]

[0014] The use of antibiotics is a common treatment for bacterial infections, and they are often also prescribed for viral infections. This prolific use has come under criticism for various reasons, most notably for inducing microbial resistance to previously effective antibiotics and rendering them less effective or ineffective against dangerous human pathogens. Administration of broad- Atorney Docket No. 225003-701601 spectrum antibiotics also has a profound effect on the normal flora and can result in colonization with antibiotic-resistant organisms. Antibiotic-mediated disruption of the normal flora can lead to fungal infections, such as invasive candidiasis, or to antibiotic-associated colitis caused by Clostridium difficile. Systems and methods comprising the non-invasive validated, redox potential measuring device, (ROXmeter) as described herein can be used to determine a disruption in normal flora and to detect a colonization of antibiotic-resistant organisms.

[0019]

[0015] Provided herein are methods for identifying a presence or a risk of one or more conditions in a subject, wherein the one or more conditions correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject, the methods comprising (a) obtaining a fecal sample of the subject; (b) contacting the fecal sample with an oxidation-redox potential (ORP) measuring device for a time sufficient to measure an ORP value of the fecal sample; and (c) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates the presence of the one or more conditions; a reference ORP value in a range of from about -100 mV to about -250 mV indicates the risk of the one or more conditions; and a reference ORP value of less than about -250 mV indicates an absence of the one or more conditions, thereby determining the presence or the risk, or the absence of the one or more conditions in the subject. Also provided herein are methods, wherein the contacting (i.e., taking of measurements) is performed within 10 minutes after the fecal sample is produced by the subject. Also provided herein are methods, wherein the contacting is performed within 5 minutes after the fecal sample is produced by the subject. Also provided herein are methods, wherein the contacting is performed within 2 minutes after the fecal sample is produced by the subject. Also provided herein are methods, wherein the contacting is maintained for a duration of from 2 seconds to 120 seconds. Also provided herein are methods, wherein the duration comprises a series of contacting at 2 second intervals for a duration of the contacting. Also provided herein are methods, wherein the one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes, hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof.

[0020]

[0016] Provided herein are ex vivo methods for identifying a presence or a risk of one or more conditions in a subject, wherein the one or more conditions correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject, the methods comprising: (a) contacting an electrode system with a first buffer solution under oxidizing conditions, wherein the electrode system is connected to an oxidation-reduction potential (ORP) measuring device; (b) contacting the electrode system with a second buffer solution under reducing conditions; (c) contacting the Atorney Docket No. 225003-701601 electrode system with a biological sample non-invasively produced by subject in a time sufficient to measure an ORP value of the biological sample; and (d) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates the presence of the one or more conditions; a reference ORP value in a range of from about -100 mV to about - 250 mV indicates the risk of the one or more conditions; and a reference ORP value of less than about -250 mV indicates an absence of the one or more conditions, thereby determining the presence or the risk, or the absence of the one or more conditions in the subject. Also provided herein are ex vivo methods, wherein the electrode system comprises a working electrode and a reference electrode. Also provided herein are ex vivo methods, wherein the working electrode comprises a planar carbon electrode, a platinum electrode, or a combination thereof. Also provided herein are ex vivo methods, wherein the reference electrode comprises a silver-silver chloride electrode. Also provided herein are ex vivo methods, wherein the electrode system further comprises a counter electrode. Also provided herein are ex vivo methods, wherein the first buffer solution comprises ferricyanide ions in a phosphate buffer at a redox potential around +350 mV. Also provided herein are ex vivo methods, wherein the second buffer solution comprises cysteine in a phosphate buffer at a redox potential around -250 mV. Also provided herein are ex vivo methods, wherein the biological sample comprises a fecal sample.

[0021]

[0017] Provided herein are methods of diagnosing a presence or predicting a risk of one or more conditions in a subject, wherein the one or more conditions correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject, the method comprising: (a) obtaining a fecal sample of the subject; (b) contacting the fecal sample with an oxidation-redox potential (ORP) measuring device to obtain an ORP value of the fecal sample; and (c) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates the presence of the one or more conditions; a reference ORP value in a range of from about -100 mV to about - 250 mV indicates the risk of the one or more conditions; and a reference ORP value of less than about -250 mV indicates an absence of the one or more conditions, thereby diagnosing the presence or the risk, or the absence of the one or more conditions in the subject. Also provided herein are methods of diagnosis, wherein the one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes (T2D), hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof.

[0022]

[0018] Provided herein are ex vivo methods for identifying an effectiveness of a therapeutic intervention for treating one or more conditions in a subject, wherein the one or more conditions Atorney Docket No. 225003-701601 correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject, comprising: (a) measuring a first redox potential of a first biological sample non-invasively produced by the subject before administering the therapeutic intervention; (b) measuring a second redox potential of a second biological sample non-invasively produced by the subject after administering the therapeutic intervention; and (c) comparing the first redox potential and the second redox potential, wherein a decrease in the redox potential after the therapeutic intervention correlates to positive effect of the therapeutic intervention. Also provided herein are methods of diagnosis, wherein the one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes (T2D), hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof.

[0023]

[0019] Provided herein are methods comprising determining a presence of one or more fatty acidproducing bacteria in a gut microbiome of a subject comprising: (a) obtaining a fecal sample of the subject; (b) contacting the fecal sample with an oxidation-redox potential (ORP) measuring device to obtain an ORP value of the fecal sample; and (c) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates an absence of the one or more fatty acid-producing bacteria in the gut microbiome of the subject; and a reference ORP value of less than about -250 mV indicates the presence of the one or more fatty acidproducing bacteria in the gut microbiome of the subject, thereby identifying a need for increasing the presence of the one or more fatty acid-producing bacteria in the gut microbiome of the subject when the ORP value is more than about -250 mV. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises a butyrate-producing bacteria, a lactate-producing bacteria, an acetate-producing bacteria, a propionate-producing bacteria, or any combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Firmicutes. Also provided herein are methods, wherein one or more fatty acidproducing bacteria comprises Clostridial Clusters IV and XIVa. Also provided herein are methods, wherein one or more fatty acid producing bacteria comprises Lachnospiraceae, Ruminococcaceae, Veillonellaceae, Acidaminococcaceae, Erysipelotrichaceae , Verrucomicrobiaceae. or any combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Lachnospira spp, Butyricicoccus spp, Eubacterium spp, Roseburia spp, Anaerostipes spp, Coprococcus spp, Faecalibacterium spp, Subdoligranulum spp, Dialisterspp, Phascolarctobacterium spp, or any combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Butyricicoccus pullicaecorum, Atorney Docket No. 225003-701601

[0024] Agathobacter rectalis (formerly Eubacterium rectale), Roseburia inulinivorans, Roseburia intestinalis, Roseburia faecis, Roseburia hominis, Roseburia cecicola, Anaerobutyricum hallii (formerly Eubacterium hallii), Anaerobutyricum soehngenii, Anaerostipes hadrus, Anaerostipes butyraticus, Anaerostipes caccae, Coprococcus eutactus, Blautia obeum, Coprococcus catus, Faecalibacterium prausnitzii, Subdoligranulum variabile, Dialister invisus, Phascolarctobacterium succinatutens, Eubacterium biforme, Akkermansia muciniphila, or any combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Coprococcus, Roseburia, Bifidobacterium, Faecalibacterium prausnitzii and Akkermansia muciniphila,' or a combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Faecalibacterium prausnitzii.

[0025]

[0020] Provided herein are methods for detecting a dysbiosis in a subject, wherein the dysbiosis correlates with an oxidation-redox potential (ORP) of a fecal sample of a subject, the method comprising: (a) obtaining a fecal sample of the subject; (b) contacting the fecal sample with an oxidation-redox potential (ORP) measuring device to obtain an ORP value of the fecal sample; and (c) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates a presence of the dysbiosis in the subject; and a reference ORP value of less than about -250 mV indicates an absence of the dysbiosis in the subject, thereby identifying a need for treating the dysbiosis of the subject when the ORP value is more than about -250 mV. Also provided herein are methods, wherein the dysbiosis is treated by modulating the presence of one or more fatty acid-producing bacteria in the gut microbiome of the subject. Also provided herein are methods, wherein the one or more fatty acid-producing bacteria comprises a butyrate-producing bacteria, a lactate-producing bacteria, an acetate-producing bacteria, a propionate-producing bacteria, or any combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Firmicutes. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Clostridial Clusters IV and XlVa. Also provided herein are methods, wherein one or more fatty acid producing bacteria comprises Lachnospiraceae, Ruminococcaceae, Veillonellaceae, Acidaminococcaceae, Erysipelotrichaceae, Verrucomicrobiaceae , or any combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Lachnospira spp, Butyricicoccus spp, Eubacterium spp, Roseburia spp, Anaerostipes spp, Coprococcus spp, Faecalibacterium spp, Subdoligranulum spp, Dialisterspp , Phascolarctobacterium spp, or any combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Butyricicoccus pullicaecorum, Agathobacter rectalis (formerly Eubacterium rectale), Roseburia inulinivorans, Roseburia intestinalis, Roseburia faecis, Roseburia hominis, Atorney Docket No. 225003-701601

[0026] Roseburia cecicola, Anaerobutyricum hallii (formerly Eubacterium halin'), Anaerobutyricum soehngenii, Anaerostipes hadrus, Anaerostipes butyraticus, Anaerostipes caccae, Coprococcus eutactus, Blautia obeum, Coprococcus catus, Faecalibacterium prausnitzii, Subdoligranulum variabde, Dialister invisus, Phascolarctobacterium succinatutens, Eubacterium biforme, Akkermansia muciniphila, or any combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Coprococcus, Roseburia, Bifidobacterium, Faecalibacterium prausnitzii and Akkermansia muciniphila, or a combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Faecalibacterium prausnitzii .

[0027]

[0021] Provided herein are methods for identifying whether a subject is a responder or nonresponders to a dietary intervention, the method comprising: (a) obtaining a fecal sample voided by the subject; (b) contacting the fecal sample with an oxidation-redox potential (ORP) measuring device to obtain an ORP value of the fecal sample; and (c) correlating the ORP value to a response or a non-response to the dietary intervention.

[0028]

[0022] Provided herein are systems for identifying a presence or a risk of one or more conditions in a subject, wherein the one or more conditions correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject, the system comprising: (a) an electrode system connected to an oxidation-reduction potential (ORP) measuring device; (b) one or more buffer solutions for calibrating the electrode system; and (c) instructions for operating the electrode system and ORP measuring device for obtaining an ORP value from a biological sample of a subject.

[0029]

[0023] Provided herein are kits for determining a subject’s risk for having or developing one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes (T2D), hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof, the kit comprising, (a) an oxidation-reduction potential (ORP) measuring device; (b) an electrode system; and (c) instructions for determining an ORP value of a biological sample non- invasively obtained from the subject; wherein the ORP value correlates to the presence of the one or more conditions when the ORP value is above than -100 mV; the ORP value correlates to the risk of the one or more conditions when the ORP value is between -300 mV and -100 mV; and the ORP value correlates to an absence of the one or more conditions when ORP value is below - 300 mV. Also provided herein are kits, wherein the electrode system comprises a working electrode, a reference electrode, a counter electrode, or a combination of thereof. Also provided herein are kits, further comprising an oxidizing solution comprising a ferricyanide ions in a Atorney Docket No. 225003-701601 phosphate buffer at a redox potential of +350 mV. Also provided herein are kits, further comprising a reducing solution comprising a cysteine in a phosphate buffer at a redox potential of -250 mV. Also provided herein are kits, further comprising a pH sensor for measuring a pH of the biological sample. Also provided herein are kits, wherein the one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes (T2D), hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof.

[0030]

[0024] Provided herein are devices comprising an oxidation-reduction potential (ORP) measuring component coupled to an electrode system, wherein the device has an ejection activator to eject the electrode system after use. Also provided herein are devices, wherein the electrode system comprises a working electrode, a reference electrode, a counter electrode, or a combination of thereof. Also provided herein are devices, wherein the electrode system is bio-fouling resistant. Also provided herein are devices, wherein the device further comprises a pH sensor.

[0031] INCORPORATION BY REFERENCE

[0032]

[0025] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034]

[0026] The novel features of the disclosure are set forth with particularity in the appended claims. An improved understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0027] FIG. 1 shows an exemplary electrical cell for generating a gut redox potential by employing riboflavin as a redox mediator and coupling a microbial redox reaction to the anode, and circuit diagram.

[0035]

[0028] FIG. 2 shows an exemplary redox meter prototype with a platinum working electrode and an Ag / AgCl reference electrode.

[0036]

[0029] FIG. 3 is a graph showing measurement of redox potential at different positions of a sample of human feces.

[0037]

[0030] FIG. 4 shows comparative redox potential analysis of feces samples from germ-free (GF) and conventionally raised (CVR) mice. Atorney Docket No. 225003-701601

[0038]

[0031] FIG. 5 is a graph showing sample fecal redox potential profiles using an OG Carbon electrode.

[0039]

[0032] FIG. 6 is a graph showing sample fecal redox potential profiles using OG Carbon electrode between different individuals and compared to a sample stored at room temperature for 12 h.

[0040]

[0033] FIG. 7 depicts a study design for a proposed clinical protocol trial described herein.

[0041]

[0034] FIGS. 8A-8C show pictures of an exemplary oxidation-reduction potential (ORP) measuring device of the present disclosure, as shown from a front view (FIG. 8A), from the front view with an LCD device showing values as detected (FIG. 8B), and a back view of the device, showing an ejector button (FIG. 8C).

[0042] DETAILED DESCRIPTION

[0043]

[0035] The present disclosure relates to exploring of a redox activity of a gut microbiome and its implications for health and diseases of mammalian subjects.

[0044]

[0036] The gut environment is strictly anaerobic where redox potentials are often below -300 mV, supporting growth of strict anaerobic gut microbes. However, within the human gut mucosa, microbes may encounter transient oxygen exposures at certain levels, which could prove lethal under in vitro conditions. In the past, a simple “gas tube” test tube was developed to resolve the paradox of an oxygenated gut mucosal environment, enabling growth of strictly anaerobic bacteria under conditions typically considered detrimental for them. As a proof of concept, F. prausnitzii was used as a model microbe, as it is one of the most abundant gut microbes and a key butyrate producer with beneficial properties. For the first time, Applicants demonstrated that the extremely oxygen-sensitive (EOS) bacterium F. prausnitzii exhibits electrogenic properties, producing an electric current, defined as the flow of electrons. Moreover, this bacterium is capable of transferring electrons to oxygen through the riboflavin-thiol shuttle. Riboflavin, also known as vitamin B2, acts as a cofactor for a wide variety of cellular processes and plays a key role in maintaining health in humans. However, bacteria are able to exploit extracellular electron transfer by variety of mechanisms such as redox active proteins and c-type cytochromes. Altogether, these metabolic activities generate a redox potential within the human gut. The application of the redox potential in understanding gut microbial metabolism, physiology and ecology is not limited to human gut.

[0045]

[0037] Metabolism, the process fundamental to life, relies on a complex mechanism of electron disposal to generate energy. Under aerobic conditions, oxygen acts as a final electron acceptor. Conversely, under anaerobic conditions, electrons are predominantly disposed of through the production of short-chain fatty acids and organic acids like lactate, acetate, propionate, and Atorney Docket No. 225003-701601 butyrate as shown in FIG. 1, where a theoretical insight of generating gut redox potential by employing riboflavin as redox mediator was demonstrated using an electrical cell to couple microbial redox reactions to the anode.

[0046]

[0038] The gut microbes possess a variety of redox active enzymes, proteins, and cytochromes that could facilitate electron transfer to alternative electron acceptors such as nitrates and sulfates. Importantly, these processes occur at more positive redox potentials. Gut microbiota is fundamental in maintaining gut redox potential, as evident from the comparative redox potentials of the cecum in germ-free mice versus conventionally raised mice, where germ-free mice present positive redox potential as compared to the conventionally raised mice.

[0047]

[0039] Applicants developed a prototype device that is composed of a platinum working and a silver / silver chloride reference electrode, as shown in FIG. 2. Samples of human feces show different redox potential at various positions using the device of the present application (FIG. 3).

[0040] The present disclosure provides methods for identifying a presence or a risk of one or more conditions in a subject. In some embodiments, the methods comprise obtaining a fecal sample of the subject. In some embodiments, the fecal sample is contacted with an oxidation-redox potential (ORP) measuring device. In some embodiments, the fecal sample is contacted within 10 minutes after the fecal sample is produced by the subject. In some embodiments, the fecal sample is contacted within 9 minutes after the fecal sample is produced by the subject. In some embodiments, the fecal sample is contacted within 8 minutes after the fecal sample is produced by the subject. In some embodiments, the fecal sample is contacted within 7 minutes after the fecal sample is produced by the subject. In some embodiments, the fecal sample is contacted within 6 minutes after the fecal sample is produced by the subject. In some embodiments, the fecal sample is contacted within 5 minutes after the fecal sample is produced by the subject. In some embodiments, the fecal sample is contacted within 4 minutes after the fecal sample is produced by the subject. In some embodiments, the fecal sample is contacted within 3 minutes after the fecal sample is produced by the subject. In some embodiments, the fecal sample is contacted within 1 minute after the fecal sample is produced by the subject.

[0048]

[0041] In some embodiments, the contacting is for a time sufficient to measure an ORP value of the fecal sample. In some embodiments, the contacting is maintained for a duration of from 2 seconds to 120 seconds. In some embodiments, the contacting is maintained for a duration of about 2 seconds, about 4 seconds, about 6 seconds, about 8 seconds, about 10 seconds, about 12 seconds, about 14 seconds, about 16 seconds, about 18 seconds, about 20 seconds, about 22 seconds, about 24 seconds, about 26 seconds, about 28 seconds, about 30 seconds, about 32 seconds, about 34 seconds, about 36 seconds, about 38 seconds, about 40 seconds, about 42 seconds, about 44 Attorney Docket No. 225003-701601 seconds, about 46 seconds, about 48 seconds, about 50 seconds, about 52 seconds, about 54 seconds, about 56 seconds, about 58 seconds, about 60 seconds, about 62 seconds, about 64 seconds, about 66 seconds, about 68 seconds, about 70 seconds, about 72 seconds, about 74 seconds, about 76 seconds, about 78 seconds, about 80 seconds, about 82 seconds, about 84 seconds, about 86 seconds, about 88 seconds, about 90 seconds, about 92 seconds, about 94 seconds, about 96 seconds, about 98 seconds, about 100 seconds, about 102 seconds, about 104 seconds, about 106 seconds, about 108 seconds, about 110 seconds, about 112 seconds, about 114 seconds, about 116 seconds, about 118 seconds, or about 120 seconds.

[0049]

[0042] In some embodiments, the duration comprises a series of contacting at 2 second intervals for a duration of the contacting.

[0050]

[0043] In some embodiments, the methods comprise comparing the ORP value to a reference ORP value. In some embodiments, a reference ORP value of more than about -100 mV indicates the presence of the one or more conditions. In some embodiments, the presence of one or more conditions is indicated by an ORP value of more than about -102 mV, more than about -103 mV, more than about -104 mV, more than about -105 mV, more than about -106 mV, more than about -107 mV, more than about -108 mV, more than about -109 mV, more than about -110 mV, more than about -111 mV, more than about -112 mV, more than about -113 mV, more than about -114 mV, more than about -115 mV, more than about -116 mV, more than about -117 mV, more than about -118 mV, more than about -119 mV, more than about -120 mV, more than about -121 mV, more than about -122 mV, more than about -123 mV, more than about -124 mV, more than about -125 mV, more than about -126 mV, more than about -127 mV, more than about -128 mV, more than about -129 mV, more than about -130 mV, more than about -140 mV, more than about -150 mV, more than about -160 mV, more than about -170 mV, more than about -180 mV, more than about -190 mV, more than about -200 mV, more than about -210 mV, more than about -220 mV, more than about -230 mV, more than about -240 mV, more than about -250 mV, more than about -260 mV, more than about -270 mV, more than about -280 mV, more than about -290 mV, or more than about -300 mV.

[0051]

[0044] In some embodiments, a reference ORP value in a range of from about -100 mV to about -300 mV indicates the risk of the one or more conditions. In some embodiments, the risk of the one or more conditions in indicated by an ORP value of about -100 mV, about -101 mV, about - 102 mV, about -103 mV, about -104 mV, about -105 mV, about -106 mV, about -107 mV, about -108 mV, about -109 mV, about -110 mV, about -111 mV, about -112 mV, about -113 mV, about -114 mV, about -115 mV, about -116 mV, about -117 mV, about -118 mV, about -119 mV, about -120 mV, about -121 mV, about -122 mV, about -123 mV, about -124 mV, about - Atorney Docket No. 225003-701601

[0052] 125 mV, about -126 mV, about -127 mV, about -128 mV, about -129 mV, about -130 mV, about -140 mV, about -150 mV, about -160 mV, about -170 mV, about -180 mV, about -190 mV, about -200 mV, about -210 mV, about -220 mV, about -230 mV, about -240 mV, about -250 mV, about -260 mV, about -270 mV, about -280 mV, about -290 mV, or about -300 mV.

[0053]

[0045] In some embodiments, a reference ORP value of less than about -300 mV indicates an absence of the one or more conditions. In some embodiments, the absence of the one or more conditions is indicated by an ORP value of less than about -130 mV, less than about -131 mV, less than about -132 mV, less than about -133 mV, less than about -134 mV, less than about - 135 mV, less than about -136 mV, less than about -137 mV, less than about -138 mV, less than about -139 mV, less than about -140 mV, less than about -141 mV, less than about -142 mV, less than about -143 mV, less than about -144 mV, less than about -145 mV, less than about -146 mV, less than about -147 mV, less than about -148 mV, less than about -149 mV, less than about -150 mV, less than about -151 mV, less than about -152 mV, less than about -153 mV, less than about -154 mV, less than about -155 mV, less than about -156 mV, less than about -157 mV, less than about -158 mV, less than about -159 mV, less than about -160 mV, less than about -170 mV, less than about -180 mV, less than about -190 mV, less than about -200 mV, less than about -210 mV, less than about -220 mV, less than about -230 mV, less than about -240 mV, less than about -250 mV, less than about -260 mV, less than about -270 mV, less than about -280 mV, less than about -290 mV, or less than about -300 mV.

[0054]

[0046] In some embodiments, there are provided methods, wherein the one or more conditions correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject. In some embodiments, the one or more conditions comprises a metabolic disorder. In some embodiments, the one or more conditions comprises an infectious disorder. In some embodiments, the one or more conditions comprises an inflammatory disorder. In some embodiments, the inflammatory disorder is a systemic inflammation. In some embodiments, the inflammatory disorder is an inflammation of the GI system of a subject. In some embodiments, the inflammatory disorder is an inflammation of a nervous system of a subject. In some embodiments, the one or more conditions comprises a dysbiosis in the gut microbiome. In some embodiments, the dysbiosis in the gut microbiome correlates with an oxidation-redox potential (ORP) of a fecal sample of a subject. In some embodiments, the dysbiosis is characterized by a reduction or an absence of bacteria associated with the microbiome of a healthy individual. In some embodiments, the one or more conditions comprises obesity. In some embodiments, the one or more conditions comprises metabolic dysfunction associated steatotic liver disease (MASLD). In some embodiments, the one or more conditions comprises type 2 diabetes (T2D). In some embodiments, the one or more Atorney Docket No. 225003-701601 conditions comprises hyperlipidemia. In some embodiments, the one or more conditions comprises hypertension. In some embodiments, the one or more conditions comprises cardiovascular disease. In some embodiments, the one or more conditions comprises a gut specific condition. In some embodiments, the one or more conditions comprises irritable bowel syndrome. In some embodiments, the one or more conditions comprises an inflammatory bowel disease. In some embodiments, the one or more conditions comprises ulcerative colitis. In some embodiments, the one or more conditions comprises Crohn's disease. In some embodiments, the one or more conditions comprises a Clostridium difficile infection. In some embodiments, the one or more conditions comprises any combination of the foregoing. In some embodiments, the one or more conditions is not limited to the listed diseases.

[0055]

[0047] In some embodiments, there is provided ex vivo methods for identifying a presence or a risk of one or more conditions in a subject. In some embodiments, the method comprises an electrode system. In some embodiments, the electrode system is connected to an ORP measuring device In some embodiments, the ex vivo methods comprise contacting an electrode system with a first buffer solution. In some embodiments, the first buffer solution is under oxidizing conditions. In some embodiments, the first buffer solution comprises ferricyanide ions. In some embodiments, the first buffer solution is a phosphate buffer solution. In some embodiments, the first buffer solution is at a redox potential around +350 mV.

[0056]

[0048] In some embodiments, the ex vivo methods comprise contacting the electrode system with a second buffer solution. In some embodiments, the second buffer solution is under reducing conditions. In some embodiments, the second buffer solution comprises cysteine. In some embodiments, the second buffer solution is a phosphate buffer. In some embodiments, the second buffer solution is at a redox potential around -250 mV.

[0057]

[0049] In some embodiments, the ex vivo methods further comprise contacting the electrode system with a biological sample. In some embodiments, the biological sample is non-invasively produced by subject. In some embodiments, the contacting is for a time sufficient to measure an ORP value of the biological sample. In some embodiments, the ex vivo methods further comprise comparing the ORP value to a reference ORP value. In some embodiments, a reference ORP value of more than about

[0058] -100 mV indicates the presence of the one or more conditions. In some embodiments, a reference ORP value in a range of from about -100 mV to about -250 mV indicates the risk of the one or more conditions. In some embodiments, a reference ORP value of less than about -250 mV indicates an absence of the one or more conditions. Atorney Docket No. 225003-701601

[0059]

[0050] In some embodiments of the present disclosure of the ex vivo methods, the electrode system comprises a working electrode. In some embodiments, the electrode system comprises a reference electrode. In some embodiments, the working electrode comprises a planar carbon electrode. In some embodiments, the working electrode comprises a platinum electrode. In some embodiments, the working electrode comprises a combination of a planar carbon electrode and a platinum electrode. In some embodiments, the reference electrode comprises a silver-silver chloride electrode.

[0060]

[0051] In some embodiments, of the ex vivo methods of the present disclosure, the electrode system further comprises a counter electrode.

[0061]

[0052] In some embodiments, of the ex vivo methods of the present disclosure, the biological sample comprises a fecal sample.

[0062]

[0053] Some embodiments of the present disclosure provide for methods of diagnosing a presence or a risk of one or more conditions in a subject.

[0063]

[0054] Some embodiments of the present disclosure provide for ex vivo methods for identifying an effectiveness of a therapeutic intervention for treating one or more conditions in a subject. In some embodiments, the ex vivo methods comprise measuring a first redox potential of a first biological sample non-invasively produced by the subject before administering the therapeutic intervention. In some embodiments, the ex vivo methods further comprise measuring a second redox potential of a second biological sample non-invasively produced by the subject after administering the therapeutic intervention. In some embodiments, the ex vivo methods further comprise comparing the first redox potential and the second redox potential. In some embodiments, a decrease in the redox potential after the therapeutic intervention correlates to positive effect of the therapeutic intervention.

[0064]

[0055] Some embodiments of the present disclosure provide for methods of diagnosis, wherein the one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes (T2D), hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof.

[0065]

[0056] Some embodiments of the present disclosure provide for methods for determining a presence of one or more fatty acid-producing bacteria in a gut microbiome of a subject. In some embodiments, the methods comprise obtaining a fecal sample of the subject. In some embodiments, the fecal sample is contacted with an ORP measuring device to obtain an ORP value of the fecal sample. In some embodiments, the method further comprises comparing the ORP value Atorney Docket No. 225003-701601 to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates an absence of the one or more fatty acid-producing bacteria in the gut microbiome of the subject; and a reference ORP value of less than about -250 mV indicates the presence of the one or more fatty acid-producing bacteria in the gut microbiome of the subject, thereby identifying a need for increasing the presence of the one or more fatty acid-producing bacteria in the gut microbiome of the subject when the ORP value is more than about -100 mV.

[0066]

[0057] In some embodiments, the one or more fatty acid-producing bacteria comprises a butyrate- producing bacteria. In some embodiments, the one or more fatty acid-producing bacteria comprises a lactate-producing bacteria. In some embodiments, the one or more fatty acid-producing bacteria comprises an acetate-producing bacteria. In some embodiments, the one or more fatty acidproducing bacteria comprises a propionate-producing bacteria. In some embodiments, the one or more fatty acid-producing bacteria comprises any combination of the foregoing.

[0067]

[0058] In some embodiments, the one or more fatty acid-producing bacteria comprises Firmicutes. In some embodiments, the one or more fatty acid-producing bacteria comprises Clostridial Clusters IV and XlVa. In some embodiments, the one or more fatty acid producing bacteria comprises Lachnospiraceae, Ruminococcaceae, Veillonellaceae, Acidaminococcaceae, Erysipelotrichaceae , Verrucomicrobiaceae. or any combination thereof. In some embodiments, the one or more fatty acid-producing bacteria comprises Lachnospira spp, Butyricicoccus spp, Eubacterium spp, Roseburia spp, Anaerostipes spp, Coprococcus spp, Faecalibacterium spp, Subdoligranulum spp, Dialister spp, Phascolarctobacterium spp, or any combination thereof. In some embodiments, the one or more fatty acid-producing bacteria comprises Butyricicoccus pullicaecorum, Agathobacter rectalis (formerly Eubacterium rectale), Roseburia inulinivorans, Roseburia intestinalis, Roseburia faecis, Roseburia hominis, Roseburia cecicola, Anaerobutyricum hallii (formerly Eubacterium hallii), Anaerobutyricum soehngenii, Anaerostipes hadrus, Anaerostipes butyraticus, Anaerostipes caccae, Coprococcus eutactus, Blautia obeum, Coprococcus catus, Faecalibacterium prausnitzii, Subdoligranulum variabile, Dialister invisus, Phascolarctobacterium succinatutens, Eubacterium biforme, Akkermansia muciniphila, or any combination thereof. In some embodiments, the one or more fatty acid-producing bacteria comprises Coprococcus, Roseburia, Bifidobacterium, Faecalibacterium prausnitzii and Akkermansia muciniphila, or a combination thereof. Also provided herein are methods, wherein one or more fatty acid-producing bacteria comprises Faecalibacterium prausnitzii.

[0068]

[0059] In some embodiments, the present disclosure provides methods for detecting a dysbiosis in a subject, the methods comprising: (a) obtaining a fecal sample of the subject; (b) contacting the fecal sample with an ORP measuring device to obtain an ORP value of the fecal sample; and Atorney Docket No. 225003-701601

[0069] (c) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates a presence of the dysbiosis in the subject; and a reference ORP value of less than about -250 mV indicates an absence of the dysbiosis in the subject, thereby identifying a need for treating the dysbiosis of the subject when the ORP value is more than about -100 mV. In some embodiments, the dysbiosis is treated by modulating the presence of one or more fatty acid-producing bacteria in the gut microbiome of the subject.

[0070]

[0060] In some embodiments, the present disclosure provides methods for identifying whether a subject is a responder or non-responders to a dietary intervention. In some embodiments, the method comprising obtaining a fecal sample produced by the subject. In some embodiments, the method further comprises contacting the fecal sample with an ORP measuring device to obtain an ORP value of the fecal sample . In some embodiments, the method further comprises correlating the ORP value to a response or a non-response to the dietary intervention.

[0071]

[0061] In some embodiments, the present disclosure provides for identifying a presence or a risk of one or more conditions in a subject. In some embodiments, the system comprises an electrode system connected to an ORP measuring device. In some embodiments, the system further comprises one or more buffer solutions for calibrating the electrode system. In some embodiments, the system further comprises instructions for operating the electrode system and ORP measuring device for obtaining an ORP value from a biological sample of a subject.

[0072]

[0062] In some embodiments, the present disclosure provides kits for determining a subject’s risk for having or developing one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes (T2D), hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof. In some embodiments, the kit comprises an ORP measuring device. In some embodiments, the kit comprises an electrode system. In some embodiments, the kit comprises instructions for determining an ORP value of a biological sample non-invasively obtained from the subject. In some embodiments, the electrode system comprises a working electrode, a reference electrode, a counter electrode, or a combination of thereof. In some embodiments, the kit further comprises an oxidizing solution comprising a ferricyanide ions in a phosphate buffer at a redox potential of +350 mV. In some embodiments, the kit further comprises a reducing solution comprising a cysteine in a phosphate buffer at a redox potential of -250 mV. In some embodiments, the kit further comprises a pH sensor for measuring a pH of the biological sample. Atorney Docket No. 225003-701601

[0073]

[0063] In some embodiments, the present disclosure provides devices comprising an ORP measuring component coupled to an electrode system. In some embodiments, the device has an ejection activator to eject the electrode system after use. In some embodiments, the electrode system is bio-fouling resistant. In some embodiments, the devices further comprise a pH sensor.

[0074]

[0064] Measured redox potentials have been used to study microbial energetics and ecology primarily in environmental contexts such as soil, water, and sewage treatment plants and ingestible electronic technologies for advanced monitoring of the gastro-intestinal tract, in vivo wireless sensors (capsule) for gut microbiome redox monitoring also exist.

[0075]

[0065] The present application obtains results ex vivo, without using an ingestible device or an invasive procedure by measuring redox potentials of biological samples in the form of fecal samples obtained from subjects.

[0076]

[0066] In some embodiments, the device of the present application comprises a working carbon electrode. In some embodiments, the device comprises an Ag / AgCl reference electrode. In some embodiments, a voltmeter (oxidation reduction potential) measuring device of the present application logs data over variable time intervals. In some embodiments, the device comprises a planar electrode with large disc working surface area of 4 mm.

[0077]

[0067] In some embodiments, oxidation reduction potential measurements are measured via a planar screen-printed electrode. In some embodiments, a method of the present disclosure comprises insertion of an electrode in the ORP measuring device. In some embodiments, the device is activated, and an electrode is inserted in a fecal sample or other biological matrix to obtain an ORP value of the sample. In some embodiments, an automatic measuring of the ORP is obtained between a carbon working electrode and an Ag / AgCl reference electrode. In some embodiments, data obtained is logged and results are displayed in mV. In some embodiments, the electrode is then ejected from the device by activating an ejector button. In some embodiments, the electrode is customized in length. In some embodiments, the electrode is disposable in sewage.

[0068] In some embodiments, the device is integrated with a pH probe to get simultaneous values of pH and Eh.

[0078]

[0069] In some embodiments, the system of the present disclosure further comprises an electronics module to store data related to ORP measuring device. In some embodiments, the system of the present disclosure further comprises a wireless communication module.

[0079]

[0070] In some embodiments, the wireless communication module provides healthcare professionals with access to real-time data through wireless communication with external devices such as mobile phones, and wearable monitors. Atorney Docket No. 225003-701601

[0080]

[0071] In some embodiments, the wireless communication module communicates with receivers such as smartphones, and wearable monitoring, using low power radio frequency transmissions. In some embodiments, the wireless communication module comprises electronic components. In some embodiments, the electronic components comprise printed circuit boards (PCB). In some embodiments, the PCB are made of fiberglass-reinforced epoxy resin. In some embodiments, the electronic components comprises radio frequency (RF) transmitters. In some embodiments, the RF transmitters comprise transistors and capacitors.

[0081]

[0072] In some embodiments, data flow between the device of the present application and external devices comprises the wireless connection module using a strong transmission protocol. In some embodiments, to improve data integrity, and security, the protocol comprises data encryption methods and error-checking processes. In some embodiments, the data obtained from the device or system of the present application is first encoded into a format that is appropriate for wireless transmission. In some embodiments, the data is compressed to reduce the amount of bandwidth required, and the packet structure is optimized for effective transmission. In some embodiments, the device or system of the present disclosure processes and analyses measured data from one more sensors and combines information to provide extensive understanding of the gut microbiome. In some embodiments, the system of the present disclosure comprises a working electrode having a silicon surface that has immobilized thereon a redox-active moiety, wherein the redox-active moiety has an oxidation potential and / or reduction potential that is sensitive to the presence of an analyte; a counter electrode and optionally a reference electrode; a source for supplying a plurality of potentials to the working electrode; and a device for measuring current through the working electrode at the plurality of potentials.

[0082]

[0073] In some embodiments, the device or system further comprises a computation system that communicates with the device or system for measuring current, having algorithms for calculating reduction or oxidation potential from the measured current at a plurality of potentials. In some embodiments the currents measured at a plurality of potentials are used to determine analyte concentration, and the determined analyte concentration is used to control a process parameter.

[0083]

[0074] In some embodiments, the devices and systems comprise a sensor for measuring ion concentration in a biological sample, comprising: an electrode configured to be in contact with the biological sample, the electrode comprising a silicon surface that has immobilized thereon a redoxactive moiety, wherein the redox-active moiety has an oxidation potential and / or reduction potential that is sensitive to concentration of said ion.

[0084]

[0075] In some embodiments, the subject silicon surface comprises a surface modified with redoxactive functional groups. In some embodiments, at least one redox-active functional group on the Atorney Docket No. 225003-701601 surface is sensitive to the presence and or the level of a substance in the biological sample. In some embodiments, the silicon surface will have at least one redox-active functional group sensitive to an analyte, and at least one redox-active functional group that is substantially insensitive to the analyte to be tested. In some embodiments, the substantially insensitive group can act as a reference, allowing for greater accuracy and reproducibility of the measurements.

[0085]

[0076] The redox groups can be chemically or physically bound to the surface. The redox groups can be attached to the silicon covalently, can be adsorbed to the silicon, or can be attached to polymers that are either covalently or non-covalently bound to the surface. Covalent binding of either the redox group or the polymer to which the redox group is a part can be beneficial in improving the lifetime and stability of the electrode. Silicon can form covalent bonds with carbon, and thus is a desirable substrate for functionalizing with carbon based molecules. The covalent binding to the surface can be through a bond between silicon and carbon, oxygen, nitrogen, sulfur, or other atom. In some embodiments the bond is between silicon and carbon. In some embodiments the bond is between silicon and oxygen. The physical bonding can occur through adsorption, and can include, for example, spontaneous self-assembly onto the silicon surface of molecules such as those derived from fatty acids which comprise redox active moieties.

[0086]

[0077] The redox-active moieties generally have reversible redox activity with well-defined cyclic voltammetry oxidation and / or reduction peaks. A suitable reference redox reagent can vary from application to application or medium to medium depending on the intended use. The position of the reduction and / or oxidation potentials of the redox active moiety can be chosen in order to improve the accuracy and quality of the measurement of redox potential. In some cases, the reduction and / or oxidation potential can be chosen to be away from other redox active species. The silicon surface generally has a wide window in which to perform measurement of reduction or oxidation potential without interfering with the measurement of the reduction and / or oxidation of the redox active moieties bound to the surface.

[0087]

[0078] Redox-active moieties that are insensitive to the presence of analytes should show little or no change in their oxidation and / or reduction potentials in the presence or absence of such analytes.

[0079] One aspect of the invention is a system for measuring analyte concentration. In one embodiment, the system comprises: a working electrode having a silicon surface that has immobilized thereon a redox active moiety, wherein the redox active moiety has an oxidation potential and / or reduction potential that is sensitive to the presence of an analyte; a counter electrode and optionally a reference electrode; a source for supplying a plurality of potentials to the working electrode; and a device for measuring current through the working electrode at the plurality of potentials. The working electrode referred to herein can comprise the silicon Atorney Docket No. 225003-701601 electrochemical sensor described above. It is desirable in many embodiments that the silicon surface also has immobilized thereon a second redox active moiety having an oxidation potential and / or reduction potential that is insensitive to the presence of said analyte. The redox active moiety that is insensitive to the presence of the analyte can be on the same silicon surface, or can be on another surface in electrical contact with the system and in contact with the sample. The system is configured such that the working electrode, the counter electrode, and optionally the reference electrode are in contact with the sample.

[0088]

[0080] In some embodiments, the system will have two or more working electrodes. For example, in some embodiments, the system will have one working electrode comprising a silicon surface that has immobilized thereon a redox active moiety whose oxidation potential and / or reduction potential is sensitive to the presence of said analyte, and a second working electrode comprising redox active moiety whose oxidation potential and / or reduction potential is insensitive to the presence of said analyte. An example of a system with two working electrodes is a system having two silicon wafers, one of which has a redox active moiety which is sensitive to pH, such as anthracene, and another redox active moiety which is insensitive to pH, such as a ferrocene. In some embodiments, the silicon wafer on which each redox active species is immobilized will be a different type of silicon wafer. In some embodiments, the silicon wafer to which the pH sensitive moiety is bound may have one doping level, and the silicon wafer on which the pH insensitive moiety is bound may have a different doping level. This type of construction can be beneficial because, in some cases, one type of redox active species will perform better in terms of amplitude, sensitivity or stability with one type of doping, while another redox active species will perform better on a silicon wafer with a different type of doping. In some embodiments, the pH sensitive moiety, e.g., anthracene, is bound to a silicon wafer that has a low level of doping, and the pH insensitive moiety, e.g., ferrocene is bound to a silicon wafer that has a higher level of doping. In some embodiments the silicon wafer onto which the pH sensitive moiety, e.g. anthracene is bound has a resistivity between about 1 Q-cm to about 1000 Q-cm, or between about 10 Q-cm to about 90 Q-cm, or between about 10 Q-cm to about 40 Q-cm while silicon wafer onto which the pH insensitive moiety, e.g. ferrocene, is bound has a resistivity between about 0.001 Q-cm and 0.1 Q- cm, or from about 0.001-0.005 Q cm resistivity, about 0.02-0.05 Q-cm. In some embodiments, an N-type silicon wafer is used for the pH insensitive moiety, e.g., ferrocene. In some embodiments, an N-type silicon wafer is used for both the pH sensitive and the pH insensitive moiety.

[0089]

[0081] In some embodiments, the system will have 3 or more working electrodes. For example, in some embodiments, the system will have one working electrode comprising a silicon surface that has immobilized thereon a redox active moiety that is sensitive to the presence of a first Atorney Docket No. 225003-701601 analyte, a second working electrode comprising a silicon surface that has immobilized thereon a redox active moiety that is sensitive to the presence of a second analyte, and a third working electrode comprising a silicon surface that has immobilized thereon a redox active moiety that is insensitive to the presence of either the first analyte nor the second analyte. The system can also have more than 3 working electrodes, for example having 4, 5, 6, 7, 8, 9, 10, 12, 20, 50 or more working electrodes, each having redox active moieties sensitive to and analyte. These systems can also have one or more than one silicon working electrode having a redox species that is insensitive to the analytes, for example to provide a reference. In some embodiments, more than one redox species that it insensitive to the analyte can be used.

[0090]

[0082] In some embodiments, the system further comprises a computation system that communicates with the device for measuring current. The computation system can have algorithms for calculating reduction or oxidation potential from the measured current at a plurality of potentials from the voltammetry measurements. The computing systems can be part of the sensing system, in some cases allowing the sensing system to be self-contained. The computing system can comprise memory for storing raw and / or processed data from the sensors. The computing system can be connected to a transmission device that will wirelessly or by wire transmit processed data to an external device. In some embodiments, the computing system can provide signals and measurements which can be transmitted in some cases in real time, allowing the system to alert end users of conditions which may require attention.

[0091]

[0083] In some embodiments the system is made up of a housing that holds the silicon electrode sensor which is electrically connected to a unit comprising the source for supplying a plurality of potentials and the current measuring device. In some embodiments the unit also comprises the computing system described above for at least partially analyzing the data. The unit can be battery powered, or can have a connection to an outside power source. The unit can have a display and input buttons to allow the user to control the measurement and to read the output from the sensor. The unit can have transmission capability for sending out data, and for receiving instructions or to be tested by an external device.

[0092]

[0084] In some embodiments of the present disclosure, the device of the present disclosure is incorporated in a smart toilet. In some embodiments, the smart toilet is integrated with a ORP measuring device, and circuitry configured to conveniently access and automatically analyze a user's biological waste or fecal matter to determine an ORP value. In some embodiments, the smart toilet system can then process and analyze the ORP value in order to make appropriate diagnoses and / or to recommend various courses of action to the user so as to promote / preserve health as described herein in accordance with the methods of present disclosure. In some embodiments, the 1 Atorney Docket No. 225003-701601 smart toilet comprises a processor (such as a central processing unit (CPU), a microcontroller, etc.) coupled to a computer-readable memory (e.g., a computer-readable storage medium). In some embodiments, the processor executes computer-executable instructions stored on the memory, thereby executing or facilitating execution of ORP measurements. In some embodiments, the computer-executable components comprises a filtration component that can automatically (e.g., not requiring manual handling by the user) collect, filter, and / or prepare a fecal sample of a user from a toilet. In some embodiments, the filtration component collects the fecal sample actively, such as by a pump and / or propeller that, in response to execution by the processor, actively draws in the fecal sample from the toilet, or by a mechanical manipulator and / or end effector that, in response to execution by the processor, physically manipulates the user's fecal sample (e.g., grabbing, slicing, scooping, catching, obtaining, acquiring, and so on). In some embodiments, the filtration component collects the sample passively, such as by inlet and / or outlet apertures that receives and / or expels the fecal sample from and / or to the toilet during a flushing operation of the toilet. In some embodiments, the filtration component includes a stationary fixture and / or apparatus in which the fecal sample is naturally deposited and / or separated as the sample enters the toilet bowl and / or as the fecal sample is flushed away. In some embodiments, a combination of fixtures, structures, and / or apparatuses are incorporated into the filtration component to facilitate the automated collection of the fecal sample. In some embodiments, incorporation of the system or device of the present disclosure into a smart toilet eliminating the need to manually collect and handle fecal samples, thereby improving accessibility and convenience of microbiome screening / monitoring using the disclosed technology.

[0093] EXAMPLES

[0094] Example 1: Device and electrode configuration

[0095] Construction of a microbial fuel cell

[0096]

[0085] A custom-made two-chambered, microbial fuel cell was fabricated from borosilicate glass bottles with 65 ml working volumes for the cathode and anode chambers (FIG. 1). The two compartments were separated by a CMI-7000S cation exchange membrane, using a 20 cm diameter septum.

[0097]

[0086] Graphite slabs (dimensions of 5x 1.5><0.2 cm) were used as cathode and anode. The electrodes were connected to the external circuit with insulated copper wire and the bare connections were sealed with a nonconductive epoxy resin. The resistance between the wires and the electrodes was less than 2 Q. Atorney Docket No. 225003-701601

[0098]

[0087] The anode chamber contained 50 mM potassium phosphate buffer (pH 7.0) as anolyte, 0.1 M glucose and Ag / AgCl reference electrode. The cathode chamber contained 100 mM potassiumphosphate buffer (pH 7.0) with 50 mM potassium ferricyanide as catholyte. The assembled fuel cell was maintained at 37 °C and the anode and cathode chambers were purged continuously with nitrogen gas and air, respectively.

[0099]

[0088] The reduction potential of potassium ferricyanide is E° = 0.36 V, with re-oxidation by O2 (E° = 0.82 V). The bacterial oxidative metabolism was coupled to the reduction of ferricyanide using riboflavin as mediator in the anode chamber (FIG. 1).

[0100]

[0089] The data was recorded using a LabJack data acquisition system at an interval of 30 s. The OCV was recorded by operating the fuel cell in open circuit mode while the current production was determined in closed circuit mode and calculated from the voltage drop across a fixed external resistance of 150 Q (I = V / R).

[0101]

[0090] Faecalibacterium prausnitzii strain A2-165 (DSM 17677) was maintained at 37 °C on yeast extract, casitone, fatty acid and glucose (YCFAG) agar under anaerobic conditions. For the microbial fuel cell experiments, the bacterial cells were grown anaerobically in 250 ml of YCFAG broth to an optical density at 600 nm (A600) of ~0.8. Cells were harvested by centrifugation, washed in buffer (pH 7.0) and suspended in 0.5 ml of the same buffer. The suspension of resting bacterial cells obtained was used to inoculate the anode chamber of the microbial fuel cell. All buffers and media were made anaerobic by flushing, immediately after autoclaving, with oxygen- free nitrogen for 30 min, and cyclic voltammetry was performed. FIG. 2 shows a prototype device comprising a platinum wire (0.5 mm), a silver wire (0.5 mm), a multimeter.

[0102]

[0091] FIG. 3 depicts a graph showing human fecal redox potential measurements using an electrochemical workstation (CHI 660E) with a PicoAmp Booster, and using an open circuit potential (OCP) technique, with a sampling interval of 0.1 seconds and a run time of 60 seconds. 3M KC1 Agar 2%, prepared by degassing with nitrogen and solidified under anaerobic environment achieved in an anaerobic COY box. Ag wire was immersed in 100 mM HC1, for two minutes and washed with MQ water to produce an Ag / AgCl reference electrode. Platinum wires were used as working and counter electrodes. A freshly voided fecal sample was embedded in the KCl / Agar as shown in FIG. 3, and the platinum electrode was dipped, and open circuit potential (OCP) was measured via electrochemical workstation CHI600C. The OCP was regarded as redox potential and was measured at the surface (-0.5 cm) below the surface and around the center area of the fecal cylinder as shown in FIG. 3.

[0103]

[0092] In an exemplary embodiment, an OG carbon electrode was challenged under oxidizing and reducing conditions, as well as in a biological matrix, i.e., fecal matrix. In some embodiments, Attorney Docket No. 225003-701601 oxidizing and reducing conditions were produced by dissolving ferricyanide and cysteine in the phosphate buffer, respectively, as shown in Table 1, below.

[0104] Table 1: Comparative redox potential analysis of OG-carbon electrode vs Pt-Ag / AgCl

[0105]

[0093] As shown in Table 1, two or more calibration points can be used, and appropriate correction factors can be applied on raw data obtained from the device depending on the relationship between the readings.

[0106]

[0094] For example, a linear calibration equation (Y = mX + c) may be used, where X represents the raw reading from the device, Y is a calibrated value that matches the reference, m is the slope or gain, and c is an intercept or offset. When the relationship is nonlinear, a polynomial (quadratic) correction equation (Y = aX2+ bX + c) can be applied, where a, b, and c are the polynomial coefficients.

[0107] Example 2: Ex vivo methods demonstrated on samples from mice

[0108]

[0095] In an exemplary embodiment, redox potential measurements in the fecal matrices of Swiss webster mice feces were demonstrated (FIG. 4). Freshly voided fecal samples from conventionally raised mice (CVR) and germ-free (GF) mice (n=3) were collected in a container. Open circuit potential (OCP) was measured immediately after collection via an OG-C0400 (Palmsens) electrode and an electrochemical workstation CHI600C for 60 seconds and a sampling interval of 0.1 seconds.

[0109]

[0096] Without wishing to be bound to theory, germ-free mice, lacking gut microbiota, are expected to produce more positive Eh values compared to conventionally raised mice harboring complete and mature gut microbiome. Using the device of the present disclosure, consistent Eh values for these mice were observed, as shown in FIG. 4.

[0110] Example 3: Ex vivo methods demonstrated on samples from human subjects

[0111]

[0097] Redox potential measurements in real matrix (human feces) are depicted graphically (FIG. 5) using freshly voided fecal samples from 4 (n=4) healthy adult donors, which were collected in a container and processed within 5 min of defecation. Open circuit potential (OCP) was measured twice from the same sample using an OG-C0400 electrode and electrochemical workstation Atorney Docket No. 225003-701601

[0112] CHI600C for 60 seconds and a sampling interval of 0.1 seconds. The results showed comparable fecal redox potential profiles (Eh values) within the range of ±25mV.

[0113]

[0098] Redox potential measurements in real matrix (human feces) were determined in freshly voided fecal samples from three (n=3) healthy adult donors, after being collected in a container (FIG. 6) Two samples (n=2) were processed within 5 min of defecation. While one sample was stored at room temperature in a container under ambient conditions. Open circuit potential (OCP) was measured via an OG-C0400 electrode and electrochemical workstation CHI600C for 60 seconds and a sampling interval of 0.1 seconds. Samples collected from one donor and stored for 12 h aerobically showed more positive Eh values, compared to fecal redox potential profiles measured of other individuals and processed within 5 minutes after defecation.

[0114]

[0099] Fecal redox potentials in volunteer’s feces during the screening phase are shown in Table 2.

[0115] Table 2: Fecal redox potential in volunteers

[0116]

[0100] Table 2 shows fecal redox potentials in mV (raw data) obtained from the ORP measuring device of the present disclosure, measured in fresh feces by the volunteers participating in the clinical trial during the screening phase. The data presented here have not been subjected to any linear or polynomial correction factor. The data was collected at an interval of 1-3 days or the same day. Subjects 5, 8, and 9 show intraday variation in fecal redox potential while subjects 1-4, 6-8, and 10-15 data showed inter-day variation in their fecal redox potential measurements. Atorney Docket No. 225003-701601

[0117]

[0101] Without wishing to be bound to theory, the above data of the present disclosure suggests a health criteria level for oxidation reduction potential values of freshly collected feces sample to be in a range starting from -250 mV and below.

[0118]

[0102] Demography and clinical variables of selected participants are shown in Table 3 below.

[0119] Table 3: Demographic and clinic variables of participants

[0120]

[0103] Table 3 provides demographic profiles of some human subjects enrolled in the study. The group of participants comprise both male and female individuals. As shown in Table 3, subjects are classified as overweight or obese as based on their body mass index (BMI). The participant’s age distribution includes middle-aged adults and elderly individuals, and the participants shown here present with one or more underlying medical conditions and are undergoing treatment with drugs.

[0121]

[0104] The above-mentioned demographic and clinical variables as listed in Table 3 are known to influence a gut microbial composition. Alterations in gut microbiota alone, or combination with environmental factors thereafter may modulate host gut / fecal redox potential through microbial metabolic activity.

[0122]

[0105] As shown in Table 3, the redox potential exhibits variation among the participants. Subject 6 presented relatively positive fecal redox potentials of 76 mV and 46 mV on day 1 and day 3 (see Table 2). This could be linked to one or more underlying conditions such as higher BMI value of 34.2 (classified as obesity class 1), open pylorus surgery or anti-hypertensive drug therapy. Attorney Docket No. 225003-701601

[0123]

[0106] Without wishing to be bound by theory, such combinations as described herein enable a redox profiling, in order to able to foresee efficacy of an intervention, or provide a prediction or diagnosis, or be able to identify biomarkers and therapeutic targets for drug, dietary intervention, precision medicine, and / or microbiome-based gut microbial modulation across diverse populations.

[0124]

[0107] The present disclosure proposes a combination of demographic, clinical, and microbiome data to assess redox dynamics. The present disclosure may further be extended to other factors, such as behavioral and lifestyle factors.

[0125] Example 4: Clinical trial protocol

[0126]

[0108] Recent studies have reported that individuals may show different metabolic responses to the same diet, in acute and extended meal settings. Differential responses in glycemia, lipid profiles, inflammation biomarkers and appetite have been shown in response to standardized meals using muffins, test drinks and regular meals. Individuals with different response patterns may be at different risk of developing chronic diseases such as obesity, type 2 diabetes mellitus (T2D), cardiovascular disease, as well as their preconditions. Several factors have been dissected to cause the differential metabolic responses including genetic set up, gut microbiota, health conditions, anthropometries, background diet and food properties. Intensive research is ongoing to identify novel biomarkers that could reflect if a person belongs to a certain response type or risk group. Such information may have important implications for more precise and more effective early prevention of disease using tailored dietary strategies, i.e., personalized nutrition. The amounts and quality of carbohydrates in the diet have major implications for cardiometabolic health and disease. Carbohydrate quality can be indicated by whole- versus refined grains, dietary fiber content, glycemic index (GI) and amount of sugar in the diet. It is well established that GI will have an impact on acute postprandial glycemic responses in both healthy individuals and individuals with T2D. Lowering of postprandial glucose responses to meals is associated with a reduced risk of cardiovascular disease among individuals with T2D, but the link to long-term health effects is more controversial among healthy individuals or individuals with prediabetes.

[0127]

[0109] A exemplary study has suggested that long-term adoption of a personalized diet that is optimized to keep blood glucose levels steady at an individual level is more beneficial compared to a healthy Mediterranean diet when it comes to risk factors for T2D among individuals with prediabetes. The long-term metabolic effects of a low- versus a high GI diet in the context of a healthy Mediterranean diet pattern in individuals with increased risk for T2D, where a low GI diet was supported, was also investigated. However, there are large inter-individual differences in Attorney Docket No. 225003-701601 glycemic responses to standardized meals with regular foods. Two response clusters from standardized meal studies were identified that were differentially associated with risk factors for T2D, where the metabolically beneficial response cluster was associated with higher abundance of butyrate producing bacterial geneses at baseline. Another two differential response clusters were found based on data from continuous glucose monitoring that were differentially associated with cardiometabolic risk profiles. Determinants of the different response groups however remain to be revealed.

[0128]

[0110] Exemplary studies have also shown that the relative presence of specific bacterial genera, namely Prevotella and Bacteroides, is associated with differential responses to high cereal fiber interventions in terms of glycemia, lipid profile, and weight loss. In addition, the copy number of the enzyme AMY1 that is coding for salivary amylases that degrade starch influences the ability to digest starch and might also have an impact on glucose homeostasis and obesity traits. Moreover, there is a suggested beneficial interaction between presence of Prevotella and low copy number of AMY 1 for effects on body weight.

[0129] Aims of a proposed clinical trial protocol

[0130] [Hl] The overall aim with the proposed clinical trial protocol is to characterize differential metabolic responses to three types of starch meals containing slow digestible starch (SDS), resistant starch (RS), or rapid digestible starch (RDS) across individuals with different microbiota and AMY I copy number.

[0131]

[0112] Specifically, the proposed trial protocol will:

[0132]

[0113] (A) Investigate differential metabolic responses (glycemia, blood lipids and inflammation biomarkers) in response to meal tolerance tests based on SDS, RS, or RDS served for breakfast and dinner across individuals with high or low abundance of Prevotella and high or low AMY 1 copy number.

[0133]

[0114] (B) Evaluate metabolic effects of chronic consumption of SDS and RS versus RDS daily for two weeks across different enterotypes and in individuals with high or low AMY1 copy number.

[0134]

[0115] (C) Evaluate a non-invasive method to measure redox potential in fecal samples, as disclosed herein. In exploratory analyses, associations between fecal redox potential and responses to different starches as well as microbiota and other biomarkers will be investigated.

[0135]

[0116] Without wishing to be bound by theory, it is hypothesized that different starches will give rise to differences in metabolic responses, irrespective of gut microbiota and / or AMY1 enzyme copy number. Further hypothesized is that individuals with high abundance of Prevotella in their fecal microbiota at baseline will have better capacity to ferment SDS and RS that may reach their colon than individuals with low abundance of Prevotella. Moreover, it is hypothesized that the Atorney Docket No. 225003-701601 effect will be even stronger among individuals with a low copy number of AMY1 enzymes. This will be due to larger amounts of starch reaching the colon where they will be fermented, which will beneficially affect postprandial glycemia.

[0136]

[0117] Based on above, the hypothesize of the present disclosure is that response clusters can be identified, and that type of starch can be matched to response type based on a meal test and thereby provide guidance towards healthier personalized eating.

[0137]

[0118] Finally, it is hypothesized that a new device for noninvasive measurement of redox-status in fecal samples will provide biologically meaningful information.

[0138] Methods

[0139] Overview of study design

[0140]

[0119] A 12-week randomized, controlled, three-way cross-over trial will be conducted, where differential metabolic responses to three types of starch will be investigated across individuals with different microbiota and copy number of AMYl.

[0141]

[0120] All participants will be randomized to three different 17-day feeding regimes, each containing a preparation day, a meal challenge test day at clinic, a 14-day dietary intervention at home followed by an oral glucose tolerance test (OGTT) at clinic.

[0142]

[0121] Three different feeding regimes will contain three different types of starch: (A) slow digestible starch, (B) resistant starch, and (C) rapid digestible starch. Each feeding regime will be separated by a 14-day wash-out period. Blood, saliva and fecal samples will be collected during the trial, together with data from continuous glucose monitoring (CGM), anthropometric measurements, self-reported appetite, dietary intake and lifestyle, food preferences, monitoring of physical activity, and sleep, as well as questionnaires of health-related quality of life, sleep quality, and daytime sleepiness.

[0143] Participants eligibility criteria

[0144]

[0122] The eligibility criteria have been designed to select adults with increased risk for developing T2D and cardiovascular diseases. Therefore, adults aged 30-70 years with abdominal obesity (z.e., a waist circumference >102 cm for men and >88 cm for women) and a BMI between 27-40 kg / m2will be recruited. Individuals taking antihypertensive and statin drugs will be asked to keep the type and dosage unchanged through the study period, or to notify investigators in case of changes. All participants will sign an informed consent before enrolment in the study.

[0145] Participants inclusion criteria

[0146]

[0123] The inclusion criteria for the proposed clinical trial will include:

[0147] • Men and post-menopausal women; Atorney Docket No. 225003-701601

[0148] • Age 30-70 years;

[0149] • BMI 27-40 kg / m2;

[0150] • Waist circumference > 102 / 88 cm for men / women;

[0151] • Weight stable during previous 3 months (± 3 kg);

[0152] • Willingness to consume intervention diets;

[0153] • Ability to deal with monitoring devices;

[0154] • Medications stable for the previous 14 days; and

[0155] • Signed informed consent.

[0156] Participants exclusion criteria

[0157]

[0124] The exclusion criteria for the proposed clinical trial will include:

[0158] • Cardiovascular events (myocardial infarction or stroke) during the previous 6 months;

[0159] • Diagnosis of diabetes (any type) or use of any drug that can interfere with glucose homeostasis (i.e., metformin, incretin analogues, SGLT-2 inhibitors);

[0160] • History of stomach or gastrointestinal conditions (e.g., inflammatory bowel disease, Crohn’s disease, malabsorption, etc.);

[0161] • Colostomy, bowel resection, bariatric surgery or other major gastrointestinal surgery;

[0162] • Renal or liver failure (creatinine >1.7 mg / dl and alanine aminotransf erase / aspartate aminotransferase > 2 times than normal values, respectively);

[0163] • Anemia (hemoglobin below the age and sex specific normal reference ranges at screening);

[0164] • Blood donation (or participation in a clinical study with blood sampling) within 30 days prior to inclusion;

[0165] • Having been treated with antibiotics within the past 3 months or planning to undergo treatment during the study period;

[0166] • Habitual use of probiotic / prebiotic supplements or foods enriched in probiotics;

[0167] • Celiac disease;

[0168] • Vegan / vegetarian diet (or a diet incompatible with protocol diets);

[0169] • Intense physical activity regimen (>7 h / week of moderate intensity or >3 h / week of high intensity);

[0170] • History of drug or alcohol abuse;

[0171] • Any other reason for lack of suitability for participation in the trial, as judged by the principal investor or co-principal investigator;

[0172] Recruitment and screening

[0173]

[0125] Individuals who meet the inclusion criteria will be screened at a clinic. The recruitment will continue until up to 400 persons have been screened. Men and women with abdominal obesity will be invited for screening, where blood samples, a saliva sample and a stool sample will be collected along with demographic information, medical history, anthropometric measurements and Atorney Docket No. 225003-701601 information about habitual diet filled out by a food frequency and lifestyle questionnaire (FFLQ). The blood sample will include standard fasting clinical chemistry measurements (glucose, HbAlc, insulin, blood lipids, hemoglobin, liver enzymes and creatinine) along with short chain fatty acids measurements. A week after the screening visit, fecal and saliva samples will be collected at home by the participants and brought to the clinic at a second screening visit. Redox potential will be measured in the stool sample by the participants. Thereafter stool samples and saliva samples will be analyzed for Prevotella abundance and HALF 1 copy number. Gut microbiota will be analyzed with 16SrRNA-sequencing. The AMY1 copy number will be obtained from saliva using droplet digital polymerase chain reaction (ddPCR).

[0174]

[0126] From the screened individuals of 400 persons, a total of 96 individuals will be included in the study. The following strata will be created among these:

[0175] • Group 1 : High Prevotella, high AMY I copy number (n = 24 individuals)

[0176] • Group 2: High Prevotella, low AMY1 copy number (n = 24 individuals)

[0177] • Group 3 : Low Prevotella, high AMY I copy number (n = 24 individuals)

[0178] • Group 4: Low Prevotella, low AMY I copy number (n = 24 individuals)

[0179]

[0127] For groups 1 and 2, individuals with relative abundance of Prevotella belonging to the highest tertile (based on up to 400 screened individuals) will be selected. Among those, the 24 individuals with the highest AMY1 copy number will be selected for Group 1 and the 24 with the lowest AMY1 copy number will be selected for Group 2.

[0180]

[0128] Similarly for groups 3 and 4, individuals belonging to the lowest tertile of relative Prevotella abundance will be selected. Among those, the 24 individuals with the highest AMY1 copy number will be selected for Group 3 and the 24 with the lowest AMY1 copy number for Group 4.

[0181] Randomization and blinding

[0182]

[0129] When participants have been screened and have provided informed consent, they will be randomized to the order of the three different diets using block-randomization. The randomization will be performed by a person not involved in the study. Participants will not be informed about the type of starch in each diet. As the dietary information will be visible on the packaging, it will not be possible to fully blind the intervention diets for the participants. The randomization code will however remain blind for investigators until completion of analyses of a priori outcomes.

[0183] Dietary interventions

[0184]

[0130] The four groups of participants will participate in a three-way cross-over feeding study.

[0185] Each feeding regime will be held three times, each consisting of a preparation day, a meal Attorney Docket No. 225003-701601 challenge day at clinic where individuals will be provided with food A, B or C two times during the day (for breakfast followed by a standardized lunch), a 14-day dietary intervention at home and a follow up OGTT visit at clinic (FIG. 7). Each feeding regime will be separated by a 14-day wash-out period between treatments.

[0186] Overview of the study

[0187]

[0131] Up to 400 men and women with abdominal obesity will be invited for screening where a stool sample and a saliva sample will be taken along with blood samples at fasting and anthropometric measurements. The gut microbiota will be analyzed with 16SrRNA together with AMY1 copy number in the saliva for creation of four different strata based on high / low Prevotella abundance and high / low AMY1 copy number. The individuals belonging to all four groups will undergo the same three-way cross-over feeding regime containing three different types of starch (A) slow digestible starch, (B) resistant starch and (C) rapid digestible starch (FIG. 7). Each treatment will begin with a meal challenge day (MC) at clinic, where participants will consume test foods for breakfast, containing the different types of starch followed by a standardized meal. Meanwhile, fifteen blood samples will be collected over six hours along with CGM-measurements. During a fourteen-day intervention at home, the participants will be asked to continue to consume the same amount of product A, B or C as during the test day for lunch and dinner along with CGM- measurements. The at home intervention will be followed up by a home challenge (HC) and a 14- day wash out period between each treatment,

[0188] Meal challenge day preparations

[0189]

[0132] The day before the meal challenge day, each participant will be provided with a blinded CGM-device and a blinded multisensory tracker of physical activity and sleep in the morning at the clinic. A saliva sample and a stool sample will also be collected during the days before the clinical visit and fecal redox potential will be recorded by the participants. The evening meal before the meal challenge day will be standardized for all participants. Participants will then also be instructed not to eat or drink anything (except for small amounts of water) from 10 pm of the evening before the meal challenge day at the clinic, and to refrain from vigorous physical activity and alcohol during the 24 hours before the clinical visit. Furthermore, participants will be instructed to avoid drinking coffee and tea during the test days at the clinic, and to keep their habitual coffee and tea intake during the whole intervention period.

[0190] Meal challenge testing day

[0191]

[0133] During the meal challenge day at the clinic, participants will be served a test breakfast and a standardized lunch. Participants will be randomized to the order of the three different feeding Atorney Docket No. 225003-701601 regimes containing three types of starch: (A) SDS, (B) RS, and (C) RDS. After arriving at the clinic, anthropometric measures (including waist circumference) will be recorded. Participants will then have 15 minutes of rest before starting with the blood sampling. Double baseline fasting blood samples will be collected at the timepoints (TP) of 15 minutes and 5 minutes before starting the meal challenge test. At TP 0, a breakfast test meal will be served, containing 80 g of available carbohydrates via different types of starch. Participants will have 15 minutes to complete the breakfast test meal and 250 ml of water will be served as part of the meal. Blood and saliva samples will be collected after the breakfast test meal, from TP +15 min and then at the TP +30, +45, +60, +90 +120, +180, and +240 min. Saliva samples will also be collected at timepoints -10, +60, +120, and +240.

[0192]

[0134] Four hours after the breakfast (TP +240), a standardized lunch with low amount of available carbohydrates will be served, which will be the same for all participants. After the lunch meal, the same blood sampling procedure will be repeated as after the breakfast (at the TP +255, +270 +285, +300, +330, and +360). Self-reported appetite will be measured during the four hours of the blood collection using visual analogue scales (VAS) every 30 min in the first two hours after breakfast and lunch, then every 60 min. After another four hours (TP +480), a standardized dinner will be served, containing 80 g of available carbohydrates via starch A, B or C. Also, for the dinner, participants will have 15 minutes to complete the meal, which will be served together with 250 ml of water. After finishing the dinner, a standardized light snack will be offered the participants before going home for consumption at TP +720.

[0193]

[0135] During the meal challenge day participants will also meet with a nutritionist who will go through the study design in detail and the dietary guidelines for the following period at home. Participants will also receive written information about how to follow the study diets and a study diary where they will report mealtimes and sleep. Key foods will be distributed at the visit containing the different sources of starch for each period. Participants will also complete questionnaires on health-related quality of life, sleep quality and daytime sleepiness at the first visit.

[0194] Intervention specific foods

[0195]

[0136] The intervention specific foods that will be used during the meal challenge day will be following (starch content for each type described in Table 4):

[0196] • (A) Slow digestible starch - Barilla Regular Spaghetti N. 5

[0197] • (B) Resistant starch - Catelli Smart Spaghetti

[0198] • (C) Rapid digestible starch - Barilla Couscous Attorney Docket No. 225003-701601

[0199] Table 4: Starch content for the test foods (per 100 g cooked weight)

[0200]

[0137] The breakfast test meal and the dinner will both contain 80 g of starch of the same type (corresponding to 230-260 g cooked weight). In other aspects, the test meals will be similar for food A, B and C in terms of macronutrient composition and energy content. The lunch and the evening snack will be the same for all participants independent of feeding regimen. The meals are further described in Table 5.

[0201] Table 5: Meals according to feeding regimen during meal challenge day

[0202] Dietary intervention at home

[0203]

[0138] After the meal challenge day, participants will be instructed to continue to consume the test foods (starch A, B and C) in similar amounts (80 g of available carbohydrates) as during the meal challenge day, at lunch and dinner for 11 days. At the start of each intervention phase, key food items will be provided, containing the different food sources of starch for the specific test period, and a variety of sauces. Participants will be provided with instructions on how to standardize the cooking procedure and they will be asked to eat the test meals fresh directly after cooking to avoid conversion of the starch in the different products to resistant starch. Participants will also be asked to note their mealtime and about how they consumed the intervention foods during the intervention periods and if the meal was eaten cold or reheated. During the study period, participants will be instructed to keep their body weight stable. Repeated calls with a nutritionist will be scheduled every three days during the intervention at home, to adequately follow-up the dietary compliance.

[0204]

[0139] During the intervention period at home, participants will wear the CGM for another 8 days. Objective measures of physical activity and sleep will be measured using a multisensory sleep Attorney Docket No. 225003-701601 tracker during the intervention period. Participants will be instructed to maintain their habitual level of physical activity and regular sleep habits and to fill out a sleep diary during the dietary intervention at home. During the last day of each intervention, participants will be asked to complete a food record. A fecal sample and a saliva sample will also be collected on the same day.

[0205] Follow up visit, Oral glucose tolerance test

[0206]

[0140] The day after finishing the fourteen-day dietary intervention at home, participants will come back to the clinic for fasting blood samples. Also, before this visit, participants will be instructed to not eat or drink anything (except for small amounts of water) from 10 pm the evening before and to refrain from vigorous physical activity and alcohol 24 hours before the testing day. After arriving at the clinic, a similar procedure will be repeated as for the meal challenge day. Anthropometric measures (including waist circumference) will be assessed, and participants will have 15 minutes of rest before starting with the blood sampling. Participants will then be instructed how to perform the blood sampling at home by a finger prick (and at the same time a fasting sample will also be taken with this method). Thereafter, the breakfast test meal will be served, containing 80 g of available carbohydrates via the different types of starch. Participants will have 15 minutes to complete the breakfast test meal and 250 ml of water will be served as part of the meal. Blood samples will then be collected at the TP +30, +60, +120, +180 and +240 min by the participants.

[0207]

[0141] During the clinical visit, questionnaires for health-related quality of life, sleep quality and daytime sleepiness will also be filled out together with the FFLQ. After the first intervention participants will have 14 days of wash-out before they start another two cycles in the same way. A schematic for one cycle of the intervention period is presented in Table 6.

[0208] Table 6: Schematic of the intervention period for feeding regimen A, B and C Attorney Docket No. 225003-701601 aAt screening;bBefore start of the first intervention

[0209] Measurements of biological samples

[0210]

[0142] AMY1 copy number at screening will be analyzed from a saliva sample where DNA is extracted and stored at -80 °C until DNA analysis. Genotyping of AMY1 copy number variants will be performed and the copy number for the AMY1 gene will be compared to the reference gene AP3B1 as determined using ddPCR with the use of a QX200 AutoDG Droplet Digital PCR System.

[0211]

[0143] Fecal samples from screening and before and after each intervention will be analyzed using 16SrRNA-sequencing. Plasma and serum samples from the fasting panel, the meal challenge test and the OGTT will also be analyzed. To be able to fully investigate the effects of the interventions the following may be analyzed in blood samples.

[0212] Fasting panel:

[0213] • Glucose;

[0214] • HbAlc;

[0215] • Insulin;

[0216] • Blood lipids;

[0217] • Hemoglobin;

[0218] • Liver enzymes;

[0219] • Creatinine; and

[0220] • Short chain fatty acids.

[0221] Meal challenge test:

[0222] • Glucose;

[0223] • Insulin;

[0224] • C-peptide

[0225] • Triglycerides;

[0226] • C-reactive protein (CRP) and Interleukine-6 (IL-6);

[0227] • Metabolomics (a selection of 8 samples per individual and treatment);

[0228] • Short chain fatty acids and 30 other gut microbiota derived (indole) compounds);

[0229] • Gut hormones (GLP-1, GIP); and

[0230] • Zonulin.

[0231] Home challenge:

[0232] • Glucose;

[0233] • Insulin; Atorney Docket No. 225003-701601

[0234] • Free fatty acids;

[0235] • Lipid profile; and

[0236] • Short chain fatty acids.

[0237]

[0144] At screening, a maximum of 20 ml blood will be drawn. During each meal challenge day at most 130 ml will be collected. During the follow up visit for the OGTT, a maximum of 10 ml blood will be sampled. In total, at most 440 ml will be collected during the whole study during a total period of 12 weeks.

[0238] Anthropometry

[0239]

[0145] Body weight together with waist circumference will be measured at screening and before and after each intervention at the clinic (see Table 3 for schematic). All measurements will be performed in the morning after urination and before eating (wearing light clothing and without shoes). Body height will be measured only at the screening visit using a wall-mounted stadiometer to the nearest 0.5 cm. BMI will be calculated as body weight (in kilograms) divided by height (in meters) squared (kg / m2). Waist circumference will be measured to the nearest 0.5 cm in standing position at navel height. Participants will be asked to take a deep breath and the measurement will be performed after a light exhalation. The waist circumference will be measured twice and then be averaged.

[0240] Self-reported appetite

[0241]

[0146] Self-reported appetite will be measured during each 8-hour meal challenge test at the clinic. Standard visual analogue scales will be administered every 30 min the first two hours after breakfast and lunch, then every 60 min. Participants will assess subjective feelings of appetite, such as hunger, desire to eat, and fullness, by marking on a 100 mm scale with end descriptions ranging from “Not at all” to “Extremely.”

[0242] Continuous glucose monitoring (CGM)

[0243]

[0147] A CGM-device will be used to obtain 24-hour continuous interstitial glucose concentrations during the meal challenge day and the dietary intervention at home. The sensor will be applied on the back of the upper part of the non-dominant arm by a study nurse during the first day of each intervention period. The sensor will be removed at the visit for the OGTT. The following CGM-measures have been considered appropriate for a population without diabetes:

[0244] • mean glucose;

[0245] • time in range (TIR);

[0246] • time above range (TAR);

[0247] • time below range (TBR);

[0248] • coefficient of variation (CV); Attorney Docket No. 225003-701601

[0249] • standard deviation (SD);

[0250] • mean amplitude of glucose excursions (MAGE);

[0251] • mean absolute glucose (MAG);

[0252] • continuous overall net glycemic action (CONGA) and lability index (LI);

[0253] • high blood glucose index (HBGI); and

[0254] • low blood glucose index (LBGI).

[0255] Monitoring of physical activity and sleep

[0256]

[0148] For objective measures of physical activity and sleep, a multisensory sleep tracker will be used during the intervention period. This will be complemented with a sleep diary, where the participants will register their bedtime and wake up-time during the interventions at home. In addition, the general level of physical activity will be evaluated using the Saltin-Grimby Physical Activity Level Scale (SGPALS). This is a questionnaire designed to assess an individual’s level of physical activity during their leisure time. It categorizes people into four levels based on their activity during the pervious 12 months (“physically inactive,” “some light physical activity,” “regular physical activity,” and “regular hard physical training for competitive sports”).

[0257] Dietary assessment

[0258]

[0149] Dietary intake will be assessed at baseline and at the last day of each intervention using a food frequency and lifestyle questionnaire (FFLQ,) together with a web-based 24-hour recall, using a web-based dietary assessment that can be filled out at home. Participants will report the dietary intake during three days at baseline (two weekdays and one day during weekend) and during the last day in each intervention period.

[0259] Health-related quality of life

[0260]

[0150] To measure health-related quality of life (HRQoL) a Medical Outcomes Study 36-Item Short Form Health Survey will be administrated at baseline and at the end of each intervention. This is a 36-item questionnaire that captures eight domains of self-perceived physical and mental health during the last week. Through algorithmic transformation domain scores are calculated for the areas physical functioning, role limitations due to physical health, bodily pain, general health, vitality, social functioning, role limitations due to emotional health and mental health. These can be presented either on a 0-100 scale of arbitrary units (raw scores) or as a norm-based T-score (with a mean of 50 AU and a standard deviation of 10). A higher score indicates greater HRQoL. T-scores are also calculated for the aggregated dimensions of physical- and mental health.

[0261] Sleep quality

[0262]

[0151] For assessment of subjective sleep, the Pittsburgh Sleep Quality Index (PSQI) will be used at baseline and at the end of each intervention. This is a 19-item questionnaire that measures sleep Atorney Docket No. 225003-701601 quality and sleep disturbances during the last month. The participants will, however, be instructed to reflect on the previous two weeks. The result is presented as a global sleep score on a 0-21 scale. A higher score indicates worse sleep quality and a value > 5 is classified as “poor sleep.” For the areas subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication and daytime dysfunction, component scores can also be calculated, each on a 0-3 AU scale.

[0263] Daytime sleepiness

[0264]

[0152] To evaluate daytime sleepiness, the Epworth Sleepiness Scale (ESS) will be used at baseline and at the end of each intervention. This is an eight-item questionnaire that measures the general level of daytime sleepiness. The result is presented as an ESS-score on a 0-24 scale, where a higher score indicates greater daytime sleepiness. A value > 10 is classified as excessive daytime sleepiness.

[0265] The primary outcomes of the project will be:

[0266] • Differences in plasma propionate between strata at timepoint +360 min

[0267] Secondary outcomes the project will be:

[0268] • Differences in glucose and insulin responses between starch treatments during breakfast, lunch meals separately, for all participants;

[0269] • Differences in glucose and insulin response (from 6-hour meal challenge test) across strata within the same dietary treatment

[0270] • Differences in glucose and insulin responses between dietary treatments during breakfast meals separately (4 h) for all participants

[0271] • Differences in glucose and insulin responses during breakfast meals separately (4 h) across strata within the same dietary treatment

[0272] • Differences in 24-hour CGM-measures and in the dynamic features of the postprandial glucose response as evaluated by CGM-home sampling between starches, for all participants;

[0273] • Differences in glucose and insulin from OGTT between starches, for all participants;

[0274] • Differences in glucose and insulin / C-petide from home sampling between dietary treatments for all participants

[0275] • Differences in inflammation biomarkers (CRP) between starches, for all participants;

[0276] • Differences in blood lipids (triglycerides) between starches, for all participants;

[0277] • Differences in short chain fatty acids (from feces and plasma) between starches, for all participants;

[0278]

[0153] Exploratory measures of the project will be:

[0279] • To evaluate differences in primary and secondary outcomes across strata for Prevotella (+ / -) and with AMY1 (+ / -); Atorney Docket No. 225003-701601

[0280] • Differences in metabolomics from 8-hour meal challenge test between starches, for all participants

[0281] • Differences in gut hormones (GLP1- and GIP) from 8-hour meal challenge test between starches, for all participants;

[0282] • Differences in anthropometric measures between starches, for all participants;

[0283] • Differences in objective measures of sleep between starches, for all participants;

[0284] • Differences in sleep quality between starches, for all participants;

[0285] • Differences in daytime sleepiness between starches, for all participants;

[0286] • Differences in health-related quality of life between starches, for all participants;

[0287] • Differences in self-reported appetite between starches, for all participants;

[0288] • Associations between fecal redox potential and responses to different starches; and

[0289] • Associations between fecal redox potential, microbiota and other clinical biomarkers.

[0290] Example 6: Data collection and readout

[0291]

[0154] The various embodiments herein relate to devices, systems, and methods used to collect biological samples from subjects in a non-invasive manner, e.g., fecal samples. In some embodiments, the biological samples are measured / analyzed using methods described herein to generate data. In some embodiments, the data collected is transmitted, and / or stored for analysis and diagnosis of the subject.

[0292]

[0155] In some embodiments, the devices described collects and processes the collected data. In some embodiments, the devices include an integrated or separate transmitter to receive and transmit the data to one or more computing devices such as a mobile device and a database server. In some embodiments, the device is connected to a system that includes a database server, which receives and / or processes the data. In some embodiments, the system includes user interfaces that receive data from the database server, summarize the data visually, providing alerts to one or more stakeholders (e.g., the subject, other users, providers, family, partners, caregivers), and / or allows them to input additional monitoring data.

[0293]

[0156] In some embodiments, the system monitors a subject’s compliance to prescribed therapy, patient complications, and / or treatment efficacy. In some embodiments, a provider sets up the subject in a monitoring system by inputting information via one or more applications on one or more computing devices, mobile devices, tablet devices, and / or browser-based web access portals. In some embodiments, information input could include an identification number assigned to the subject’s device, such as the device as described herein, the subject’s name, date of birth, initial vital signs (e.g., blood pressure, temperature, weight, oxygenation level, etc.), and / or the details of a prescribed therapy including drug type, dosage, and durations of each use cycle. In some Atorney Docket No. 225003-701601 embodiments, the subject may connect the device of the present disclosure to the system after testing a biological sample, such as a fecal sample, to determine an oxidation-reduction potential of the sample. In some embodiments, the subject then connects the device to a docking station that may be configured to charge a battery of the device, upload / download data to a memory of the device or to another storage device and / or calibrate the device. In some embodiments, subject pairs the device with a separate computing device (e.g., tablet, smartphone) via Bluetooth connectivity.

[0294]

[0157] In some embodiments, the separate computing device receives and processes the data and further transmits the data to a server (e.g., database) via a wired or wireless network connection. In some embodiments, the subject is provided access to the data and / or analysis via a graphical user interface of a computing device (e.g., tablet, mobile phone, laptop, PC). In some embodiments, the system allows the subject to input additional data into the user interface such as weight, blood pressure, oxygen saturation, and energy / fatigue levels. In some embodiments, a provider (e.g., health care professional, care provider) is provided access to the subject’s data via a graphical user interface on one or more computing devices such as a browser-based web access portal. In some embodiments, the provider reviews all of the monitored data from one or more subjects.

[0295] Example 7: Exemplary device and method of use

[0296]

[0158] An exemplary embodiment of a oxidation-reduction potential (ORP) measuring device is shown in FIGS 8A-8C, that may be used in methods disclosed herein to measure ORP values in non-invasively obtained biological samples from subjects e.g., fecal samples.

[0297]

[0159] The exemplary device, comprises an instrument cup that shields an electron inserted into the device from the instrument body. A front face of the instrument body (FIG. 8A)includes a start button to begin operation of the device once the electrode has been connected with a biological sample, and an LCD display screen (see FIG. 8B) that is capable of displaying readout values, as detected on contact of the electrode with the biological sample. A back face of the device (FIG. 8C) comprises an ejector button that, on activation, ejects the inserted electrode from the device.

[0298]

[0160] In an exemplary method of using the device, a clean electrode is inserted through an opening on an instrument shield that is placed over the top of an ORP measuring device. In some embodiment, the instrument shield is made of silicone. Once the electrode is fully inserted into the ORP measuring device, the electrodes is connected with a fecal sample to be measured. Try to keep the cup as clean as possible. Care is taken to ensure that the fecal sample is not contacted Atorney Docket No. 225003-701601 with the instrument shield. However, if there is contact, the instrument shield can be removed to be washed or replaced after obtaining ORP measurements of the fecal samples.

[0299]

[0161] After the electrodes are connected with the sample, a start button on the ORP measuring device is activated to begin taking a measurement. The device is further configured to emit an audible signal, such as a beeping sound to notify a user that the ORP measuring device is activated and capable of taking measurements automatically. Measurements may be taken for a set duration, such as for about two minutes. If the start button were to be activated with an electrode being inserted in the ORP measuring device, the ORP measuring device is emit a notification signal and will display text such as “insert card” on the LCD display screen.

[0300]

[0162] Following insertion of the electrode into the biological sample, as measurements are taken from the sample, the LCD display screen will begin displaying variable readings. The LCD display screen also displays a line timer. Once a sufficient time has passed and the reading is completed, the device will make a further audible notification to notify the user that the measurement is complete. A final reading will be displayed on the LCD screen. The used electrode can then be ejected from the ORP measuring device by activating an ejector button on the back of the device and can be suitably disposed of.

[0301]

[0163] Once the used electrode is removed, the instrument shield can be removed, washed and replaced on the ORP measuring device, or replaced with a new instrument shield. The final reading displaced on the LCD display screen can be recorded, along with a date and time that the reading was taken, and the information can be saved on the ORP measuring device and / or transferred to an external device of choice.

[0302]

[0164] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives described herein may be employed in practicing embodiments of the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Docket No. 225003-701601CLAIMSWHAT IS CLAIMED IS:

1. A method for identifying a presence or a risk of one or more conditions in a subject, wherein the one or more conditions correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject, the method comprising:(a) obtaining a fecal sample of the subject;(b) contacting the fecal sample with an oxidation-redox potential (ORP) measuring device for a time sufficient to measure an ORP value of the fecal sample; and(c) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates the presence of the one or more conditions; a reference ORP value in a range of from about -100 mV to about -250 mV indicates the risk of the one or more conditions; and a reference ORP value of less than about -250 mV indicates an absence of the one or more conditions, thereby determining the presence or the risk, or the absence of the one or more conditions in the subject.

2. The method of claim 1, wherein the contacting is performed within 10 minutes after the fecal sample is produced by the subject.

3. The method of claim 1 or 2, wherein the contacting is performed within 5 minutes after the fecal sample is produced by the subject.

4. The method of any one of claims 1-3, wherein the contacting is performed within 2 minutes after the fecal sample is produced by the subject.

5. The method of any one of claims 1-4, wherein the contacting is maintained for a duration of from 2 seconds to 120 seconds.

6. The method of claim 5, wherein the duration comprises a series of contacting at 2 second intervals for a duration of the contacting.

7. The method of any one of claims 1-6, wherein the one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes (T2D), hyperlipidemia, hypertension, cardiovascular disease, a gut specificAtorney Docket No. 225003-701601 condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof.

8. An ex vivo method for identifying a presence or a risk of one or more conditions in a subject, wherein the one or more conditions correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject, the method comprising:(a) contacting an electrode system with a first buffer solution under oxidizing conditions, wherein the electrode system is connected to an ORP measuring device;(b) contacting the electrode system with a second buffer solution under reducing conditions;(c) contacting the electrode system with a biological sample non-invasively produced by subject in a time sufficient to measure an ORP value of the biological sample; and(d) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates the presence of the one or more conditions; a reference ORP value in a range of from about -100 mV to about -250 mV indicates the risk of the one or more conditions; and a reference ORP value of less than about -250 mV indicates an absence of the one or more conditions, thereby determining the presence or the risk, or the absence of the one or more conditions in the subject.

9. The ex vivo method of claim 8, wherein the electrode system comprises a working electrode and a reference electrode.

10. The ex vivo method of claim 9, wherein the working electrode comprises a planar carbon electrode, a platinum electrode, or a combination thereof.

11. The ex vivo method of claim 9, wherein the reference electrode comprises a silver-silver chloride electrode.

12. The ex vivo method of any one of claims 8-11, wherein the electrode system further comprises a counter electrode.

13. The ex vivo method of any one of claims 8-12, wherein the first buffer solution comprises ferricyanide ions in a phosphate buffer at a redox potential around +350 mV.Atorney Docket No. 225003-70160114. The ex vivo method of any one of claims 8-13, wherein the second buffer solution comprises cysteine in a phosphate buffer at a redox potential around - 250 mV.

15. The ex vivo method of any one of claims 8-14, wherein the biological sample comprises a fecal sample.

16. A method of diagnosing a presence or a risk of one or more conditions in a subj ect, wherein the one or more conditions correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject, the method comprising:(a) obtaining a fecal sample of the subject;(b) contacting the fecal sample with an oxidation-redox potential (ORP) measuring device to obtain an ORP value of the fecal sample; and(c) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates the presence of the one or more conditions; a reference ORP value in a range of from about -100 mV to about -250 mV indicates the risk of the one or more conditions; and a reference ORP value of less than about -250 mV indicates an absence of the one or more conditions, thereby diagnosing the presence or the risk, or the absence of the one or more conditions in the subject.

17. The method of claim 16, wherein the one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes (T2D), hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof.

18. An ex vivo method for identifying an effectiveness of a therapeutic intervention for treating one or more conditions in a subject, wherein the one or more conditions correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject, comprising:(a) measuring a first redox potential of a first biological sample non-invasively produced by the subject before administering the therapeutic intervention;(b) measuring a second redox potential of a second biological sample non-invasively produced by the subject after administering the therapeutic intervention; and(c) comparing the first redox potential and the second redox potential,Atorney Docket No. 225003-701601 wherein a decrease in the redox potential after the therapeutic intervention correlates to positive effect of the therapeutic intervention.

19. The ex vivo method of claim 18, wherein the one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes (T2D), hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof.

20. A method comprising determining a presence of one or more fatty acid-producing bacteria in a gut microbiome of a subject comprising:(a) obtaining a fecal sample of the subject;(b) contacting the fecal sample with an ORP measuring device to obtain an ORP value of the fecal sample; and(c) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates an absence of the one or more fatty acid-producing bacteria in the gut microbiome of the subject; and a reference ORP value of less than about -250 mV indicates the presence of the one or more fatty acid-producing bacteria in the gut microbiome of the subject, thereby identifying a need for increasing the presence of the one or more fatty acidproducing bacteria in the gut microbiome of the subject when the ORP value is more than about -100 mV.

21. The method of claim 20, wherein one or more fatty acid-producing bacteria comprises a butyrate-producing bacteria, a lactate-producing bacteria, an acetate-producing bacteria, a propionate-producing bacteria, or any combination thereof.

22. The method of claim 20 or 21 , wherein one or more fatty acid-producing bacteria comprises Firmicutes.

23. The method of any one of claims 20-22, wherein one or more fatty acid-producing bacteria comprises Clostridial Clusters IV and XlVa.

24. The method of any one of claims 20-23, wherein one or more fatty acid producing bacteria comprises Lachnospiraceae. Ruminococcaceae. Veillonellaceae. Acidaminococcaceae. Erysipelotrichaceae, Verrucomicrobiaceae. or any combination thereof.Atorney Docket No. 225003-70160125. The method of any one of claims 20-24, wherein one or more fatty acid-producing bacteria comprises Lachnospira spp, Butyricicoccus spp, Eubacterium spp, Roseburia spp, Anaerostipes spp, Coprococcus spp, Faecalibacterium spp, Subdoligranulum spp, Dialister spp, Phascolarctobacterium spp, or any combination thereof.

26. The method of any one of claims 20-25, wherein one or more fatty acid-producing bacteria comprises Butyricicoccus pullicaecorum, Agathobacter rectalis (formerly Eubacterium rectale), Roseburia inulinivorans, Roseburia intestinalis, Roseburia faecis, Roseburia hominis, Roseburia cecicola, Anaerobutyricum hallii (formerly Eubacterium hallii), Anaerobutyricum soehngenii, Anaerostipes hadrus, Anaerostipes butyraticus, Anaerostipes caccae, Coprococcus eutactus, Blautia obeum, Coprococcus catus, Faecalibacterium prausnitzii, Subdoligranulum variabile, Dialister invisus, Phascolarctobacterium succinatutens, Eubacterium biforme, Akkermansia muciniphila, or any combination thereof.

27. The method of any one of claims 20-26, wherein one or more fatty acid-producing bacteria comprises Coprococcus, Roseburia, Bifidobacterium, Faecalibacterium prausnitzii and Akkermansia muciniphila, or a combination thereof.

28. The method of any one of claims 20-27, wherein one or more fatty acid-producing bacteria comprises Faecalibacterium prausnitzii.

29. A method for detecting a dysbiosis in a subject, wherein the dysbiosis correlates with an oxidation-redox potential (ORP) of a fecal sample of a subject, the method comprising:(a) obtaining a fecal sample of the subject;(b) contacting the fecal sample with an ORP measuring device to obtain an ORP value of the fecal sample; and(c) comparing the ORP value to a reference ORP value, wherein a reference ORP value of more than about -100 mV indicates a presence of the dysbiosis in the subject; and a reference ORP value of less than about -250 mV indicates an absence of the dysbiosis in the subject, thereby identifying a need for treating the dysbiosis of the subject when the ORP value is more than about -250 mV.

30. The method of claim 29, wherein the dysbiosis is treated by modulating the presence of one or more fatty acid-producing bacteria in a gut microbiome of the subject.Atorney Docket No. 225003-70160131. The method of claim 30, wherein the one or more fatty acid-producing bacteria comprises a butyrate-producing bacteria, a lactate-producing bacteria, an acetate-producing bacteria, a propionate-producing bacteria, or any combination thereof.

32. The method of claim 30 or 31, wherein one or more fatty acid-producing bacteria comprises Firmicutes.

33. The method of any one of claims 30-32, wherein one or more fatty acid-producing bacteria comprises Clostridial Clusters IV and XlVa.

34. The method of any one of claims 30-33, wherein one or more fatty acid producing bacteria comprises Lachnospiraceae, Ruminococcaceae, Veillonellaceae, Acidaminococcaceae, Erysipelotrichaceae, Verrucomicrobiaceae. or any combination thereof.

35. The method of any one of claims 30-34, wherein one or more fatty acid-producing bacteria comprises Lachnospira spp, Butyricicoccus spp, Eubacterium spp, Roseburia spp, Anaerostipes spp, Coprococcus spp, Faecalibacterium spp, Subdoligranulum spp, Dialisterspp , Phascolarctobacterium spp, or any combination thereof.

36. The method of any one of claims 30-35, wherein one or more fatty acid-producing bacteria comprises Butyricicoccus pullicaecorum, Agathobacter rectalis (formerly Eubacterium rectale), Anaerobutyricum soehngenii, Roseburia inulinivorans, Roseburia intestinalis, Roseburia faecis, Roseburia hominis, Roseburia cecicola, Eubacterium hallii, Anaerostipes hadrus, Anaerostipes butyraticus, Anaerostipes caccae, Coprococcus eutactus, Blautia obeum, Coprococcus catus, Faecalibacterium prausnitzii, Subdoligranulum variabile, Dialister invisus, Phascolarctobacterium succinatutens, Eubacterium biforme, Akkermansia muciniphila, or any combination thereof.

37. The method of any one of claims 30-36, wherein one or more fatty acid-producing bacteria comprises Coprococcus, Roseburia, Bifidobacterium, Faecalibacterium prausnitzii and Akkermansia muciniphila, or a combination thereof.

38. The method of any one of claims 30-37, wherein one or more fatty acid-producing bacteria comprises Faecalibacterium prausnitzii.

39. A method for identifying whether a subject is a responder or non-responders to a dietary intervention, the method comprising:(a) obtaining a fecal sample voided by the subject;Atorney Docket No. 225003-701601(b) contacting the fecal sample with an oxidation-redox potential (ORP) measuring device to obtain an ORP value of the fecal sample; and(c) correlating the ORP value to a response or a non-response to the dietary intervention.

40. A system for identifying a presence or a risk of one or more conditions in a subject, wherein the one or more conditions correlate with an oxidation-redox potential (ORP) of a fecal sample of a subject, the system comprising:(a) an electrode system connected to an oxidation-reduction potential (ORP) measuring device;(b) one or more buffer solutions for calibrating the electrode system; and(c) instructions for operating the electrode system and ORP measuring device for obtaining an ORP value from a biological sample of a subject.

41. A kit for determining a subject’s risk for having or developing one or more conditions comprising a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes, hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof, the kit comprising,(a) an ORP measuring device;(b) an electrode system; and(c) instructions for determining an ORP value of a biological sample non-invasively obtained from the subject; wherein the ORP value correlates to a presence of the one or more conditions when the ORP value is above than -100 mV; the ORP value correlates to the risk of the one or more conditions when the ORP value is between -250 mV and 100 mV; and the ORP value correlates to an absence of the one or more conditions when ORP value is below -250 mV.

42. The kit of claim 41, wherein the electrode system comprises a working electrode, a reference electrode, a counter electrode, or a combination of thereof.

43. The kit of claim 41 or 42, further comprising an oxidizing solution comprising ferricyanide ions in a phosphate buffer at a redox potential of +350 mV.

44. The kit of any one of claims 41-43, further comprising a reducing solution comprising a cysteine in a phosphate buffer at a redox potential of -250 mV.Atorney Docket No. 225003-70160145. The kit of on any one of claims 41-44, further comprising a pH sensor for measuring a pH of the biological sample.

46. The kit of any one of claims 41-45, wherein the one or more conditions comprises a metabolic disorder, obesity, metabolic dysfunction associated steatotic liver disease (MASLD), type 2 diabetes (T2D), hyperlipidemia, hypertension, cardiovascular disease, a gut specific condition, irritable bowel syndrome, an inflammatory bowel disease, ulcerative colitis, Crohn's disease, a Clostridium difficile infection, or any combination thereof.

47. A device comprising: an ORP measuring component coupled to an electrode system, wherein the device has an ejection activator to eject the electrode system after use.

48. The device of claim 47, wherein the electrode system comprises a working electrode, a reference electrode, a counter electrode, or a combination of thereof.

49. The device of claim 47 or 48, wherein the electrode system is bio-fouling resistant.

50. The device of any one of claims 47-49, wherein the device further comprises a pH sensor.