WO2023178051A1 - System and method for dejecta enzyme activity detection - Google Patents

Abstract

A system and method for dejecta enzyme activity detection to predict, mitigate and/or prevent peristomal skin complications (PSC) includes a collecting unit, an enzymatic activity detection device, and an inlet valve. The collecting unit can collect dejecta entering an ostomy bag from a stoma. The enzymatic activity detection device may include an assay unit having on or more assay chambers. The assay chambers may include an assay solution for detecting a target proteolytic enzyme. The inlet valve may be a one-way valve that transmits the dejecta from the collecting unit to the enzymatic activity detection device. The enzymatic activity detection device may further include a waste unit and analyzing unit.

Description

TITLE

SYSTEM AND METHOD FOR DEJECTA ENZYME ACTIVITY DETECTION

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is based upon and claims priority to Provisional Application No. 63/319,456 filed on March 14, 2022, the content of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] The present disclosure pertains to a system and method for dejecta enzyme activity detection for measuring biomarkers that can indicate peristomal skin complications (PSC) for an ostomy system. More particularly, the present disclosure pertains to a system and method for ostomy output (dejecta) characterization and/or PSC mitigation, indication, and/or diagnosis in ileostomy patients using enzyme assay technology.

[0003] A stoma, or type of opening in the abdomen, that diverts the small intestine, large intestine, or urinary tract, so that it protrudes from the body wall. Diversions of the small intestine, large intestine, or ureters are called ileostomy, colostomy, or urostomy, respectively. Ileostomies are an effective treatment for inflammatory intestinal disease, but the creation of a stoma and use of ostomy containment appliances can potentially pose associated health risks. One of these risks is the development of PSC, skin issues that occur around the stoma.

[0004] These complications can range from irritation and redness to more serious issues such as abscesses or fistulas. They can be caused by a variety of factors, such as leakage of dejecta, improper stoma or peristomal skin care or underlying medical conditions. Symptoms of PSC include sensations of itching and burning, irritation around the stoma, and skin erosion. Individuals suffering from PSC require treatment and care from a wound ostomy continence (WOC) nurse, but up to 80% of patients suffering from PSCs do not seek medical attention due to a failure to detect or correctly identify the condition.

[0005] A common cause of PSC is the exposure of the skin to enzymatically active dejecta. Proteolytic, lipolytic and carbohydrate-cleaving enzymes released by the pancreas or from the lumen of the small intestine can digest and corrode the skin, leading to irritation and degradation. Therefore, being able to detect the quantity of different enzymes exiting the stoma over time and developing a model for the relationship between enzyme activity and PSC severity would allow for improved diagnosis, treatment, and possible prevention of PSC conditions. Characterization of the enzymatic properties of the dejecta could inform the development of improved ostomy products, services and therapies.

[0006] Current methods of proteolytic enzyme detection involve benchtop assays where labs dilute effluent samples and carry out reactions using well defined substrates that produce a measurable product. Measurement of light intensity using a spectrophotometer can be used to calculate an overall enzyme concentration relative to a standard curve. This process provides a convenient colorimetric readout for enzyme activity, but its sensitivity is relatively low and it cannot easily be completed directly by a patient in the comfort of their home. Enzymatic activity is current measured using colorimetric or fluorescent substrates that upon enzymatic cleavage change color or release light in response to excitation, respectively. Both assays require the collection, processing and transportation of dejecta to a laboratory setting, which increases time, labor and cost as well as limits high throughput analyses.

[0007] Accordingly, there is a need to create a point-of-care device that can reliably characterize and monitor enzyme concentration and activity in dejecta and potentially predict conditions that could lead to PSC and deliver relief and reassurance to the patient in the comfort of their home.

BRIEF SUMMARY

[0008] A system and method for dejecta enzyme activity detection for an ostomy system is provided according to various embodiments.

[0009] In one aspect, a system for enzymatic activity detection is provided. The system may include a collecting unit. The collecting unit may collect dejecta entering an ostomy bag from a stoma. The system may also include an enzymatic activity detection device. The enzymatic activity detection device may include an assay unit. The assay unit may include one or more assay chambers having an assay solution for detecting a target proteolytic enzyme. The dejecta enzyme activity detection may further include an inlet valve. The inlet valve may be a one-way valve that transmits the dejecta from the collecting unit to the dejecta enzyme activity detection. The device may further include a waste unit and analyzing unit.

[0010] In an embodiment, the assay solution may include a solution optimized for a fluorescence output based on a target enzyme concentration.

[0011] In an embodiment, the assay solution may include a substrate that releases fluorescence upon cleavage. For example, the substrate can be a colorimetric or fluorescent substrate assay or protease activity.

[0012] In an embodiment, the assay solution may include fluorophore and quencher- conjugated target proteins with specific cleavage motifs for detecting the target proteolytic enzyme.

[0013] In an embodiment, the assay unit may include a light source that provides light at an excitation wavelength to the solution. [0014] In an embodiment, the assay unit may include a fluorometric sensor that measures fluorescent signals to detect the target proteolytic enzyme activity or all the proteolytic enzymes in the dejecta. In such an embodiment, the fluorometric sensor may include a complementary metal-oxide semiconductor (CMOS) sensor.

[0015] In an embodiment, the at least one assay chamber may include a first assay chamber configured to detect a first proteolytic enzyme and a second assay chamber configured to detect a second proteolytic enzyme. The first and second proteolytic enzymes may be different. In an embodiment, the assay unit may include more than two assay chambers that detect more than two proteolytic enzymes.

[0016] In an embodiment, the enzymatic activity detection device may include an inlet oneway valve that closes once a pressure gradient is met. The pressure gradient may indicate a filling of fluid of the assay unit. In such an embodiment, the enzymatic activity detection device may include a waste unit for collecting analyzed dejecta.

[0017] In an embodiment, the enzymatic activity detection device may include an analyzing unit that outputs detection information based on detected target proteolytic enzyme.

[0018] In a second aspect of the present disclosure, a method for detecting enzymatic activity is provided. The method may be applied using a system for enzymatic activity detection disclosed in an embodiment. The method may obtain dejecta from a stoma. The method may also detect a target proteolytic enzyme based on the dejecta and an assay solution. The method may further output detection information based on the detected target proteolytic enzyme.

[0019] In an embodiment, the assay solution of the method may include a solution optimized for a fluorescence output based on a target enzyme concentration.

[0020] In an embodiment, the assay solution of the method may include a red fluorescence ENZCHEK Protease assay substrate or other fluorescent or colorimetric substrate for proteases. [0021] In an embodiment, the assay solution of the method may include fluorophore and quencher-conjugated target proteins with specific cleavage motifs for detecting the target proteolytic enzyme.

[0022] In an embodiment, the method may measure fluorescent signals based on the dejecta and the assay solution. The method may further detect the target proteolytic enzyme based on the fluorescent signals.

[0023] In an embodiment, the method may detect a first target proteolytic enzyme based on the dejecta and a first assay solution in a first assay chamber. The method may further detect a second target proteolytic enzyme based on the dejecta and a second assay solution in a second assay chamber. The first and second target proteolytic enzymes may be different. In an embodiment, the method may include more than two assay chambers that detect more than two proteolytic enzymes.

[0024] In an embodiment, the method may dispose of the dejecta in a waste unit.

[0025] In an embodiment, the method may display detection information. The detection information may include a risk of PSC detection alert.

[0026] The foregoing general description and the following detailed description are examples only and are not restrictive of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The benefits and advantages of the present embodiments will become more readily apparent to those of ordinary skill in the relevant art after reviewing the following detailed description and accompanying drawings, wherein:

[0028] FIG. 1 is a schematic view of an enzymatic activity detection system with an ostomy system, according to an embodiment.

[0029] FIG. 2 is a front view of an enzymatic activity detection system and ostomy system attached to a user, according to an embodiment.

[0030] FIG. 3 is a schematic view of an enzymatic activity detection system, according to an embodiment.

[0031] FIG. 4 is an illustration of a prior art quantum dot fluorescence resonance energy transfer (FRET) reporter system.

[0032] FIG. 5 A is an illustration of the components of a prior art FRET-CCD (charged-coupled device) microchip casing device.

[0033] FIG. 5B is an illustration of the prior art FRET-CCD microchip casing of FIG. 5 A.

[0034] FIG. 6 is an illustration of a prior art colorimetric enzymatic assay.

[0035] FIG. 7A is a graph of the result from a first bottom red fluorescence ENZCHEK experiment.

[0036] FIG. 7B is a graph of the result from a first top red fluorescence ENZCHEK experiment.

[0037] FIG. 7C is a graph of the result from a second bottom red fluorescence ENZCHEK experiment.

[0038] FIG. 7D is a graph of the result from a second top red fluorescence ENZCHEK experiment.

[0039] FIG. 8A is a graph of a bottom enzyme activity assay of 500 ug/mL trypsin.

[0040] FIG. 8B is a graph of a top enzyme activity assay of 500 ug/mL trypsin.

[0041] FIG. 9A is a graph showing bottom results of the ENZCHEK assay at dilute concentrations of trypsin. [0042] FTG. 9B is a graph showing top results of the ENZCHEK assay at dilute concentrations of trypsin.

[0043] FIG. 10 is a graph showing a compared results standard for the ENZCHEK assay of FIGS. 9A and 9B.

[0044] FIG. 11 is an exploded perspective view of a detection sensor for an enzymatic activity detection device, according to an embodiment.

[0045] FIG. 12 is a wireframe view of an enzymatic activity detection device, according to an embodiment.

[0046] FIG. 13 A is an image of a test sample with a detected enzyme.

[0047] FIG. 13B is an image of a test sample without a detected enzyme.

[0048] FIG. 14 is a flow diagram illustrating a method for detecting enzymatic activity, according to an embodiment.

[0049] FIG. 15 is a schematic illustration of a computing environment for an enzymatic activity detection device, according to an embodiment.

DETAILED DESCRIPTION

[0050] While the present disclosure is susceptible of embodiment in various forms, there is shown in the drawings and will hereinafter be described presently preferred embodiments with the understanding that the present disclosure is to be considered an exemplification and is not intended to limit the disclosure to the specific embodiments illustrated. The words “a” or “an” are to be taken to include both the singular and the plural. Conversely, any reference to plural items shall, where appropriate, include the singular. The words “first,” “second,” “third,” and the like may be used in the present disclosure to describe various information, such information should not be limited to these words. These words are only used to distinguish one category of information from another. The directional words “top,” “bottom,” up,” “down,” front,” “back,” and the like are used for purposes of illustration and as such, are not limiting. Depending on the context, the word “if’ as used herein may be interpreted as “when” or “upon” or “in response to determining.”

[0051] The present disclosure provides a system and method for enzymatic activity detection for an ostomy system. The enzymatic activity detection system and method can be configured to detect proteolytic enzymes for predicting, detecting, mitigating, and/or preventing peristomal skin complications (PSC). The enzymatic activity detection system and method can provide multiple benefits to the user. For example, the system and method can assist in maintaining a user’s skin health by detecting elevated enzymatic activity, which can lead to PSC or other skin health complications. The enzymatic activity detection system and method may be applied to an ostomy barrier of a one-piece pouch system or a faceplate of a two-piece pouch system.

[0052] FIG. 1 illustrates an enzymatic activity detection system 10 with an ostomy system. According to example embodiments shown schematically in FIG. 1, the ostomy system can be a two-piece pouch system that can generally include an ostomy bag 12 and an ostomy barrier appliance 14. The ostomy bag 12 can include an ostomy barrier coupling member 18 for mounting to a user around a stoma. The ostomy barrier appliance 14 can include a coupling member 20 for mounting to the ostomy barrier coupling member 18. The enzymatic activity detection system 10 can include an enzymatic activity detection device 16 that can include a tube 22 and collecting unit 24 for collecting fluid samples from the ostomy bag 12 for analysis.

[0053] In an embodiment, the enzymatic activity detection system 10 can have dejecta or gastrointestinal fluid pumped out of the ileostomy, and the materials used to perform the enzymatic activity detection can be cycled through the enzymatic activity detection device 16 and can be recycled or disposed. To maintain homeostasis, a high uniformity of materials that are in direct contact with the gastrointestinal fluid is preferred. Tn an embodiment, elements of the enzymatic activity detection system 10 that are in direct contact with the gastrointestinal fluid can be formed of a synthetic polysulfone material in a disclosed embodiment. For example, the tube 22 can be made out of synthetic poly sulfone material. In an embodiment, sections of a poly sulfone could be augmented with glass fibers to increase strength.

[0054] FIG. 2 illustrates the enzymatic activity detection system 10 and an ostomy system attached to a user. According to the example embodiments shown schematically in FIG. 2, the ostomy barrier appliance 14 can be attached to the user, the ostomy bag 12 can be mounted to the ostomy barrier appliance 14, an end of the tube 22 (it will be appreciated that multiple tubes 22 may be attached to collecting unit 24 to allow gastrointestinal fluid to be collected from the ileostomy and delivered to the PSC detection device 16) can be attached to the ostomy barrier appliance 14, and the enzymatic activity detection device 16 can be attached to the user with, for example, an adhesive patch. In another embodiment, the enzymatic activity detection device 16 can be mounted on the ostomy bag 12 or a user’s belt, clothing or pocket.

[0055] In an embodiment, the PSC detection system 10 may further include a mobile software application on a mobile electronic device in communication with the enzymatic activity detection device 16. The enzymatic activity detection device 16 may be provided as an accessory for an ostomy system. The enzymatic activity detection system 10 can include a collecting unit 24 for collecting fluid samples for analysis by the enzymatic activity detection device 16. The enzymatic activity detection device 16 can communicate enzymatic activity detection information to a mobile application on a mobile electronic device (not shown).

[0056] FIG. 3 illustrates the enzymatic activity detection system 10. According to example embodiments shown schematically in FIG. 3, the PSC detection system 10 can generally include the enzymatic activity detection device 16, the tube 22, and the collecting unit 24. The enzymatic activity detection device 16 can include a valve 26, an assay unit 17 having one or more assay chambers 28, 30, 32, 34, a waste unit 36, and an analyzing unit 38. The enzymatic activity detection device 16 can further include a pump and pressure sensor.

[0057] In an embodiment of the enzymatic activity detection system 10, the collecting unit 24 can be a soft flexible tube that can collect fluid from the stoma as it enters the ostomy bag 12. The tube may be constructed of a synthetic polysulfone material, as discussed above, or another suitable material. In another embodiment, the collecting unit 24 can be part of a suction device that sucks fluid from around the stoma or the ostomy bag 12 through an additional tube. The collecting unit 24 can include a stoma drainage tube. For example, the stoma drainage tube can be a peripheral tubing that collects fluid from inside the ostomy bag. The collecting unit 24 can include a fdter for filtering out solid substances. It will be appreciated that filtering of the gastrointestinal fluid can decrease noise in the pressure and temperature measurements taken and avoid clogs in the system components. The filter also can filter cells passing into the enzymatic activity detection device 16. While such cells can maintain high homogeneity between the stomal output and the gastrointestinal fluid, they also increase the probability of breaches and errors. If such cells can be excluded from the gastrointestinal fluid in the enzymatic activity detection device 16 without affecting the concentration of enzymes of interest and the markers of activity thereof, a filter with pores smaller than the cells in ileum fluid should be used. Filters can be replaced with some frequency, so the enzymatic activity detection device 16 can be designed to allow for easy filter exchange.

[0058] In an embodiment of the enzymatic activity detection system 10, the tube 22 can include a cylindrical tube that can carry fluid from the collecting unit 24 to the enzymatic activity detection device 16. [0059] In an embodiment of the enzymatic activity detection system 10, the assay unit 17 can be an assay chip that can include one or more assay chambers 28, 30, 32, 34 and a sensor (See FIGS. 5A and 5B) as further discussed below.

[0060] In an embodiment of the enzymatic activity detection system 10, the tube 22 can feed into the valve 26. The valve 26 can be a one-way inlet valve that can equally feed filtered fluid into parallel assay chambers 28, 30, 32, 34 of the assay unit 17. The one-way valve 26 can include a solenoid valve that can drive the hydraulic circuitry of the enzymatic activity detection device 16. Solenoid valves can have diverse mechanisms and material composition, which is an advantage because if the tubing material is robust enough, it can also be employed in the solenoid valves.

[0061] High uniformity of the materials in direct contact with the gastrointestinal fluid can help to maintain homeostasis and reduce the opportunity for breaches at the interfaces between disparate materials. Therefore, in a disclosed embodiment, the tubing, valves, and assay chambers can be made out of one high-performance plastic, including synthetic polysulfone material, as discussed above. For the purpose of providing the pressure to drive gastrointestinal fluid through the enzymatic activity detection device 16, peristaltic pumps can be employed for high-volume fluids and syringe pumps for low-volume fluids. It will be appreciated that a slight pressure difference can occur between the stoma output and the air in the ostomy bag, which can necessitate slight additional force to drive the gastrointestinal fluid, though it may be extremely viscous. An inline impeller pump, which operates by rotating a turbine and thus driving the fluid outward, can be used in such a situation.

[0062] In an embodiment of the enzymatic activity detection system 10, faults in the hydraulic components of the enzymatic activity detection system 10 can be sensed and reported by the system. These faults can be detectable via a pressure sensor, as a breach in the hydraulics should manifest as a sharp deviation in system pressure. Oxygen saturation and factors specific to gastrointestinal fluid or the assays used to detect the enzymatic activity also may be monitored. To that end, sampling speeds should be chosen dependent on necessary response times. In other words, samples should be taken at a frequency high enough that potential anomalies are timely identified and reported.

[0063] In an embodiment of the enzymatic activity detection system 10, pressure sensing can be performed both up- and down- stream. Additionally, an air trap sensor may be used to detect bubbles in the gastrointestinal fluid. If the enzymatic activity detection system 10 releases processed gastrointestinal fluid into an ileostomy bag, air contamination from the output end of the enzymatic activity detection device 16 can be considered and engineered against. In some embodiments, temperature sensors may be used to monitor heated and cooled liquid components of the enzymatic activity detection system 10, assay reagents functional at room temperature could be used.

[0064] In an embodiment of the enzymatic activity detection system 10, saline can be used to rinse the enzymatic activity detection device 16 components of impurities after a detection cycle, however hot water may also be used depending on the components. To achieve sterilizing water temperatures, more than a standard power outlet’s voltage may be necessary. A similar circuitry architecture could be employed in cleaning cycle design.

[0065] In an embodiment where the enzymatic activity detection system 10 is mobile, low power draw can be essential AC or DC motors with variable settings could be used for high- volume pumps and DC motors for low-volume pumps. If no high-volume pumps are used, a DC power input or AC to DC converter from a suitable power source may be sufficient.

[0066] It will be appreciated that subcircuits in the enzymatic activity detection system 10 can have microcontrollers operating independently but with a uniting control system with selfmonitoring and checks in place to avoid problems. Alarms or indicators may be connected to this circuit. If the power to the enzymatic activity detection system 10 is interrupted during operation, the standard is to shut all lines. In an embodiment of the enzymatic activity detection system 10, the electrical components and plugs are grounded and are fully isolated from fluid components.

[0067] In an embodiment, the assay chambers 28, 30, 32, 34 of the assay unit 17 can each detect individual enzymes. The used gastrointestinal fluid that is output from the assay chambers 28, 30, 32, 34 after enzyme detection can be disposed of in the waste unit 36 and detection data that can be transmitted to the analyzing unit 38.

[0068] In an embodiment of the PSC detection system 10, the overall anatomy of the enzymatic activity detection device 16 may be composed of an one-way inlet valve 26, an assay unit 17 comprising at least one, and in some embodiments a plurality of parallel assay chambers 28, 30, 32, 34 or ‘chips’ for assays, and another valve (not shown) connecting the assay chamber or chip outputs to the waste unit 36, as seen in FIG. 3.

[0069] In an embodiment of the enzymatic activity detection system 10 with the ostomy system, the gastrointestinal fluid excreting into the ostomy bag 12 can enter the inlet valve 26 (which may include a filter) and then enter the assay chambers 28, 30, 32, 34 or “chips” for assays. The inlet valve 26 can be configured to close once an appropriate pressure gradient has formed due to the filling of the assay chambers 28, 30, 32, 34 or chips. Thereafter, the assays (or enzyme detection) are performed in each assay chamber 28, 30, 32, 34 (or on the ‘chips’ for assays), each assay chamber 28, 30, 32, 34 or chip being designed to detect and quantify one of the key proteolytic enzymes or other desired bacterial markers (like Beta-glucosidase or other metabolic enzymes) that may indicate the presence of a enzymatic activity . Once all assays have been completed, the second valve would open and the used gastrointestinal fluid is released into the waste unit 36. The results from each assay are quantified by a complementary metal-oxide semiconductor (CMOS) sensor, or in another embodiment by a CCD (coupled charged device) sensor, and the fluorescent readout is converted to an output signal, which informs the viewer of the concentration of each enzyme/mark of interest in tested gastrointestinal fluid.

[0070] In an embodiment of the enzymatic activity detection system 10, the enzymatic activity detection device 16 can be small enough to hook onto the side of a user’s pants or be placed into a purse without adding significant stress on the stoma or skin, similar to an insulin pump. This simplifies the sampling process and mitigates user interaction with potentially toxic and pathogenic excreted matter in the gastrointestinal fluid. Ultimately, the enzymatic activity detection device 16 can be designed to be a precise and minimally invasive product.

[0071] The enzymatic activity detection device 16 can be utilized in-situ for the user to assess the existence of, or likelihood of contracting, a PSC without necessarily having to initially contact a medical professional. Such a device allows for more continuous and precise examination with minimal inconvenience to the user.

[0072] In one or more embodiments of the enzymatic activity detection system 10, there is provided multiple enzymatic activity detection devices which utilize novel biological assays or sensors to provide enzymatic activity readouts with improved scalability, miniaturization, and handling time. Such devices are preferably designed to minimize training for users, allowing all patients to directly monitor their gastrointestinal fluid for the presence of elevated protease activity. The enzymatic activity detection system architecture may be able to be generalized for the detection of other molecules of interest, such as pro-inflammatory cytokines and bacterial markers. [0073] It will be appreciated that there are several different types of enzyme assays known in the art, including spectrophotometric assays, radiometric assays, and colorimetric assays. In a spectrophotometric assay, the enzyme reaction can be monitored by measuring the change in the absorption of light at a specific wavelength. In a radiometric assay, the enzyme reaction can be monitored by measuring the radiation emitted during the reaction. In a colorimetric assay, the enzyme reaction can be monitored by measuring the change in the color of a chemical indicator during the reaction.

[0074] In an embodiment of the enzymatic activity detection system 10, the assays in assay unit 17 can measure the activity of trypsin, chymotrypsin, carboxypeptidases, aminopeptidases, and potentially lipases. The assays depend upon the creation of unique substrates that act as specific proteolytic targets for each of the key intestinal enzymes. The target sites for each of the various intestinal enzymes are shown in Table 1 below. Due to the specificity of the individual proteolytic enzymes, select target substrates are used to perform individualized, enzyme specific assays, a mechanism that is generalizable to many different enzymes. This allows for a multichanneled assay unit, with each unit (or assay chamber) analyzing one of the specific proteolytic enzymes. Table 1 shows the target cleavage sites for a number of key small intestinal proteolytic enzymes. These unique cleavage sites form the basis of an embodiment for enzymatic assay design for an assay unit in the PSC detection system 10.

[0075] Table. 1

[0076] In an embodiment of the enzymatic activity detection system 10, the sensor construction is inspired by a FRET (Fluorescence Resonance Energy Transfer) detection system as is known in the art. FRET assays are based on manipulation of the energy transfer between two molecules through dipole-dipole interactions. More specifically, a chromophore and a quencher are placed in proximity, either linked molecularly or free floating, and light is added to the sample at a specific excitation wavelength which should lead the chromophore to emit light in return at a predictable wavelength. However, this activation is inhibited through dipole-dipole interactions with the nearby quencher. As the chromophores separate further, through processes such as cleavage, the chromophore can be activated by the excitation light and begins to emit detectable light at a predictable wavelength. Therefore, the presence of emitted light is inversely indicative of the proximity to the two interacting molecules.

[0077] In order to make the FRET system viable for the detection of proteolytic enzymes in an embodiment of the enzymatic activity detection system 10, the assay can require conjugated molecules on either side of the target enzymatic cleavage site. Therefore, in an embodiment of the enzymatic activity detection system 10, the construction of fluorophore and quencher-conjugated target proteins that contain the specific cleavage motifs for each of the enzymes of interest will be unique for each assay chamber 28, 30, 32, 34 or chip. In this way, the substrate will be covalently linked to a polymer chip, over which the gastrointestinal fluid samples will flow. Cleavage of the substrate will release the fluorophore, producing an increase in the emitted fluorescent signal, which can be measured by a sensor. Similar methods have been used to quantify enzyme activity of collagenases with substrates that are conjugated with two fluorophores to create a FRET system. [0078] One key benefit of such an assay in an embodiment of the enzymatic activity detection system 10 is the use of laminar flow throughout the system, which is easier to accomplish and control in the enzymatic activity detection system 10 than a higher rate flow. Additionally, as this is a fluorometric assay, it is ideal for use in an embodiment of the enzymatic activity detection system 10 because sensors exist that have extraordinarily high sensitivity to light emission, allowing for enzymatic activity detection in environments with low concentrations and with high accuracy.

[0079] FIG. 4 illustrates a quantum dot FRET reporter system as is known in the art. Specifically, this schematic displays how a FRET reporter system can be designed using quantum dots linked to fluorescently labeled substrates, leading to an EL excitation-based emission response.

[0080] A powerful fluorometric sensor, such as a small coupled charged device (CCD) or complementary metal-oxide semiconductor (CMOS) sensor, can convert the light given off by the fluorophores into an electric signal. Use of a CCD sensor to detect fluorescence is known in the art as used in conjunction with an electroluminescent (EL) strip. The strip can provide the excitatory light that can be absorbed and emitted through the series of FRET fluorophores. The CCD-FRET system can house the reaction in a small microchip containing multiple wells made out of poly-methyl methacrylate (PMMA), and the chip, EL, and CCD can be combined with a lens in a small black box as is known in the art, as shown in FIGS. 5A and 5B. Each well can contain 7uL of their reactant, which can be a 12-fold reduction in reaction volume compared to their assays using a bench top plate reader, and the sensitivities of these reactions were on the same order of magnitude. The miniaturized nature and high signal level of this kind of platform make it ideal for use in a wearable device such as an embodiment of the enzymatic activity detection system 10, and the assay concept can be adapted for different substrates to monitor a broad panel of enzymes as required by an embodiment of the enzymatic activity detection system 10.

[0081] FIGS. 5A and 5B illustrate a FRET-CCD microchip casing as is known in the art. Specifically, this schematic shows an example of a prior art functionalized FRET-CCD microchip in an acrylic box casing.

[0082] Another potential assay for the detection of proteolytic enzymes is the use of pNP based substrates as is known in the art. As known in the art, there exists an assay that uses a peptide substrate conjugated with a p-nitroanilide compound for the detection of chymotrypsin activity. The cleavage of this type of pNP compounds produces a measurable color, as illustrated in FIG. 6. Printable wells on paper and a smartphone can perform and measure these kinds of reactions. Though this means of measuring light intensity is fast and convenient, and could also lend itself to the creation of a large dataset, the sensitivity of this method for detection of enzymatic activity is an order of magnitude lower than the standard colorimetric method using a plate reader. As an alternative, pNP substrates may be used with stencil printed carbon electrodes that could create an electric current in response to the creation of the p-nitrophenol redox product. The change in current during the reaction on the electrodes can be measured using square wave voltammetry resulting in a sensitivity of the electrochemical method on the same order as that of a colorimetric assay using abenchtop plate reader. This method can allow for small scale analysis and successful enzyme-specific targeting through peptide-small molecule conjugation.

[0083] FIG. 6 illustrates a colorimetric enzymatic assay as is known in the prior art. Specifically, the necessary reagents and reaction mechanism for a colorimetric enzymatic assay are shown in FIG. 6 in contrast to the electrochemical scheme, highlighting its convenient result.

Color-based assays have advantages in ease of measurement, validation, and quality control.

[0084] A fluorescence-based detection device, as is known in the art, can have a high sensitivity detection of proteolytic enzymes, and it can be a novel assay concept that can be generalized to a variety of enzymes. Additionally, the modular chip design can accommodate other types of assays, and the device can be efficient and easy to use. However, a prior art fluorescencebased device can be expensive, especially for the fluorescent probes, and not easily scalable, and it can require a high level of technical expertise.

[0085] A paper-based detection device, as known in the art, can be easy and inexpensive to construct, can have well-characterized reaction paradigm, and can be easily scaled. However, such paper-based devices can have low sensitivity for proteolytic enzymes detection, can be limited to enzymes with activity on pNP substrates, can require extra handling to perform assay, may not be easily integrated into stoma bag, and may require separate devices for readout (spectrophotometer, phone camera, or multimeter).

[0086] In an embodiment of the enzymatic activity detection system 10, a fluorescence-based activity assay can be used for recognizing proteolytic enzymes. The cleavage site for each enzyme of interest can be well-defined and fluorophores can be conjugated to each specific substrate to be cleaved upon introduction of the enzyme. A biological assay can be developed using reagents and samples and readily-sourced fluorescent molecules.

[0087] More specifically, first, validation and optimization of the enzymatic reaction can be carried out to determine how much fluorophore is needed to detect a target enzyme and target enzyme concentration. Fluorescence output of a variety of different concentrations of enzyme and substrate can be measured The biological assays can be developed on a benchtop scale and miniaturized and adapted to a wearable device, such as enzymatic activity detection device 16. Within such a device, the fluorescent substrate can be conjugated to a polystyrene chip, so this conjugation process can be accomplished and optimized, and the assay can be recapitulated on the chip. The fluorescence output of each assay can also be optimized to match the sensitivity of a desired sensor. The size or shape of the wearable device, such as enzymatic activity detection device 16, may depend on the size constraints of the assay. Chip architecture can be designed with reusability in mind, and for semi-continuous measurements.

[0088] In an embodiment of the enzymatic activity detection system 10, ENZCHEK assays (as known in the art) can be used. For example, an ENZCHEK assay with two fluorophores (red and green), with distinct excitation and emission spectra, can be used. The red fluorescence assay can have a better resolution of 589 nm excited and 617 emitted wavelengths. The ENZCHEK protocol can be followed to test the assay. For example, FIGS. 7A-7D show results from a test using these assays for the PSC detection system 10. Due to the nature of the assay, for the concentration range evaluated (1 mg/mL to 5 ug/mL), the fluorescence would be roughly linear to the concentration, so a linear regression can be performed to determine the accuracy of the assay. [0089] FIG. 7A shows results from a first red fluorescence ENZCHEK bottom experiment. The graph shows a performed assay with dilutions with the following trypsin concentrations: 500 ug/mL, 250 ug/mL, 100 ug/mL, 50 ug/mL, 10 ug/mL, and 5 ug/mL. Results suggest a linear relationship between concentration and fluorescence, indicating a detection of trypsin. FIG. 7B shows results from a first red fluorescence ENZCHEK top experiment.

[0090] FIG. 7C shows results from a second red fluorescence ENZCHEK bottom experiment. The graph shows a performed assay with dilutions with the following trypsin concentrations: 1 mg/mL, 500 ug/mL, 250 ug/mL, 100 ug/mL, 50 ug/mL, 10 ug/mL, and 5 ug/mL.

[0091] FIG. 7D shows results from a second red fluorescence ENZCHEK top experiment. Linear regression analysis was performed and results are found in the bottom right of the graph.

[0092] In another embodiment of the enzymatic activity detection system 10, the ENZCHEK assay can be used to measure enzyme activity. This can be done by measuring the fluorescence over time. For example, the assay can be conducted using a 500 ug/mL sample of trypsin and consecutive fluorescence reactions every 15 min for 2 hours. All of these reactions can be evaluated simultaneously at 2 hours. These results, as seen in FIGS. 9A and 9B, can show a curvilinear trend typical of enzyme activity. However, further experiments can be performed at larger time scales to confirm complete Michaelis-Menten kinetics. FIG. 8A shows a bottom enzyme activity assay of 500 ug/mL trypsin. FIG. 8B shows a top enzyme activity assay of 500 ug/mL trypsin.

[0093] Further experimentation revealed that lower concentrations of trypsin can be used. For example, a dilute series of trypsin samples (1 ng/uL to 500 ng/uL) can be used. FIG. 9A shows a bottom ENZCHEK Assay at dilute concentrations of trypsin. FIG. 9B shows a top ENZCHEK Assay at dilute concentrations of trypsin. FIG. 10 shows the compared results standard for the ENZCHEK assay. This experiment showed a curvilinear trend that could be more precisely fit (using a square root model) and closely resembled the predicted trend for the green ENZCHEK assay.

[0094] In an embodiment of the enzymatic activity detection system 10, the wearable PSC detection device 16 can include a light source, excitation filter, emission filter, and sensor. The ENZCHEK protease assay can use the red fluorescence. In an embodiment, the gastrointestinal fluid can be diluted for improved accuracy of detection and analysis. For example, in some embodiments, protocols for sample preparation can include dilution steps to allow for detection to occur in samples with protease concentrations within the ng/mL range. In such an embodiment, analysis should can be performed on a biological-grade spectrophotometer and comparison of fluorescence detection between the sensor and the standard spectrophotometer can be done to validate the sensor. Additional time-dependent assays can be constructed over larger lengths to better understand the kinetics of the enzyme and meausre enzyme activity. Other key small intestinal digestive enzymes can be evaluated to ensure accuracy of detection for all enzymes of interest.

[0095] In an embodiment of the enzymatic activity detection system 10, the sensor can be the OD7670 camera that is known in the art, which can be a complementary metal-oxide semiconductor (CMOS) sensor rather than a charge-coupled device (CCD). The camera can be assembled in a circuit with a microcontroller, such as an Arduino Uno microcontroller as is known in the art. The camera can output .bmp fdes to a ‘C:/out’ folder. These fdes can roll out at a rate of about 1 every 4 seconds. Software can be developed to address potential types of artifacts that may be included in the images.

[0096] In an embodiment of the enzymatic activity detection system 10, a sensor software process can include a shortcut that starts a cmd line and a python script that causes the microcontroller script to be uploaded to the device, and then a java code to be activated, and finally for images to be read and processed interactively, so that a user can act as a final quality check, or even determine what kind of image correction may be needed.

[0097] In an embodiment of the enzymatic activity detection system 10, an initial test of the sensor can be performed by sandwiching, from the bottom up, a phone flashlight, the excitation filter, a clear bottom black-sided plate with spread-out samples of different concentrations and a blank, the emission filter, and the camera, all held together by tape (or other suitable means) and enclosed in a dark metal drawer (see FIG. 11). As part of testing, individual components can be validated, with filters combined letting through no light from a source and each of them picking up on the camera when applied to the phone flashlight in the drawer. Constant agitation of the circuit can lead to highly artifact-obscured images. Light leaking through openings in the assembly can show up as a pattern of light appearing in the captured images. A higher concentration can give a clear circle pattern and a lower concentration can yield a less distinct variant of the same.

[0098] In an embodiment of the enzymatic activity detection system 10, a plastic scaffold can be used to hold the CMOS sensor (which is sensitive to slight changes in the positioning of wiring and circuitry), align it with the emission and excitation filters, and allow for positioning around a single well in a 96-well plate for imaging. The design can include a minimal, 2-part design for rapid initial testing. The dimensions of the CMOS sensor, heights and diameters of the filters, and the diameter of the camera can be taken into account.

[0099] FIGS. 11-13B show testing devices 100, 200 and testing results (excited light 300) of an assay solution. The testing devices 100, 200 can generally include a similar structure that can include a light source, sensor, and assay solution chamber.

[00100] FIG. 11 shows an assembly design of an embodiment of a testing device 100 for use in enzymatic activity detection device 16. As shown in FIG. 11, the testing device 100 can include a camera cover 110, top scaffold 112, chamber scaffold 114, bottom scaffold 116, and light source 118. The camera cover 110 can include a camera 120. The top scaffold can include a camera-sized insert 122, an emission fdter inlet 124, and an opening 126. The chamber scaffold can include symmetrical indentations at the top opening and bottom opening of the chamber scaffold 114, a hole 130, a top scaffold sized insert 132, a glass-sized inlet 134, and a reaction chamber 136. The hole 130 can include a 1/16 inch opening that leads to the reaction chamber 136. An identical hole 130 can be positioned at the opposite side of the chamber scaffold. The bottom scaffold 116 can include an excitation fdter insert 138 and an opening 140. The light source 118 can include a phone flashlight or other type of light source. [00101] The top scaffold 112 can be designed as a block with a tube in the middle of varying diameter (not shown). The bottom of the tube can have a smaller diameter to allow the emission fdter to sit on top of it, while the rest of the tube is just wide enough to fit the length of the camera lens to its base. The center of the tube can be offset from the edge of the box by the same distance as the camera from the chip it sits on, to snugly fit the entire module into the box. The bottom scaffold 116 can be constructed similarly to accommodate the excitation filter, and a hollow compartment was added underneath for a light source.

[00102] In an embodiment, a large 60mm x 60mm x 60mm box can be a foundation for a chamber. A 20mm deep square cutouts can be the top and bottom of the box to fit the previously created top (35.5mm x 35.5mm) and bottom (40mm x 40mm) scaffold. A hollow square chamber can be cut out going through the body of the device with dimensions of 16mm x 16mm. This chamber can be designed such that 18mm x 18mm glass coverslips can be epoxied over the opening to create completely sealed chamber. On opposite sides of this chamber, small holes can be created each with 1/16 inch diameter. These holes can be extended to the outside of the box and were given a slight taper such that the outer hole is slightly larger in diameter than the inner hole. Because the chamber will be completely sealed, these holes can be designed to provide a channel for microfluidic tubing into and out of the device for loading and washing of the device chamber. Glass microscope slides can be affixed on either side of the chamber with epoxy and a syringe and 1/16 in can be used for microtubing to show that fluid could be made to flow through the device.

[00103] FIG. 12 illustrates a wireframe view of testing device 200. The testing device 200 can include a microfluidic scaffold with an internal chamber 210 and a wire 212 for holding microfluidic samples through cutouts at top and bottom scaffolds. For example, the sample can be between the affixed blocks. The microfluidic scaffold can be compatible with the sensor and the ENZCHEK assay.

[00104] In an embodiment, a 198 uL IX Digestion Buffer and 2 uL 1 mg/mLBodipyTR-X were combined, then 100 uL of this mixture was added to 100 uL 1 mg/mL trypsin were used with the sample. The mixture and sample can be tested on a testing device 100, 200. FIGS. 13A shows an image of the excited light 300 from a well with a sample. FIGS. 13B shows a test of an empty well without an excited light. FIG. 13A compared to FIG. 13B shows that an enzyme can be detected using an assay based on the excited light 300.

[00105] In an embodiment of the enzymatic activity detection system 10, the enzymatic activity detection device 16 can use an ENZCHEK assay that can be optimized for a specific fluorescence and concentration for the best sensitivity of an enzyme detection in a gastrointestinal fluid. A microfluidic scaffold can further be utilized with a fluorometric sensor and the ENZCHEK assay. [00106] The enzymatic activity detection system lOcan provide patients with an easy, at-home test for reading enzymatic activity in their stoma output, and it can potentially increase the number of patients from whom ileostomy aspirate data can be collected. Since few patients seek medical attention, it can be difficult for clinicians to formulate connections between stoma output and enzymatic activity diagnoses. Therefore, this kind of point-of-care assay could allow for the creation of a robust data set for these clinicians to use to assist in future diagnoses of stoma complications. This enzymatic activity detection system 10 could be further functionalized with other non-enzymatic assays to detect levels of bacteria, key metabolite and salt concentrations, pH levels, and additional markers to provide an even higher dimensionality of data on the content of the ileostomy effluent. This could further elucidate mechanisms driving PSCs and associated conditions.

[00107] FIG. 14 shows method 1400 for detecting peristomal skin complications. The method

15 may be applied to a computing device such as a wearable device, mobile device, personal computer or server. The wearable device may be an ileostomy enzyme detection device, such as enzymatic activity detection device 16.

[00108] In step 1410, the wearable device can obtain dejecta from a stoma. The dejecta can include gastrointestinal fluid.

[00109] In step 1420, the wearable device can detect at least one target proteolytic enzyme based on the gastrointestinal fluid and an assay solution. For example, an assay solution can be optimized to detect the target proteolytic enzyme in the gastrointestinal fluid. The assay solution can be optimized to be used in a wearable device and a low-cost sensor.

[00110] In step 1430, the wearable device can output detection information based on the detected at least one target proteolytic enzyme. For example, an alert can be output on the wearable device (a light, sound or haptic feedback alert) to indicate to the user that a target enzyme or level of enzymatic activity has been detected and PSC may be developing or at risk of developing. In another example, the wearable device can output a communication signal to communicate to a mobile device an alert to indicate to the user that a target enzyme or level of enzymatic activity has been detected and PSC may be developing or at risk of developing.

[00111] In an embodiment, data gained in the assay can be useful to guide the development of PSC mitigating products as well as provide biomarkers to the user that may be correlated with diet, medication, activity, and/or disease progression or remission.

[00112] In an embodiment, the assay unit may include inhibitors of specific proteases. Measurement of activity in the dejecta without the contribution of a single or multiple specifically inhibited enzyme(s) can be used to measure the relative contribution of the inhibited enzyme to the total enzymatic activity. For example, the use of inhibitors in the analyzed dejecta could add another dimension to the test unit. For example, Trypsin, TPCK Treated can be used and it may involve a chromatographically purified, diafiltered, lyophilized powder that has been treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) to inhibit contaminating chymotryptic activity. In another example, Chymotrypsin, Alpha, TLCK Treated can be used and it may involve three times crystallized and treated with l-chloro-3-tosylamido-7-amino-2- heptanone (TLCK) to inhibit trypsin activity and dialyzed against 1 mM HC1 to remove autolysis products and low molecular weight contaminants. It can be supplied as a dialyzed, lyophilized powder.

[00113] FIG. 15 shows a computing system 1500 that can be part of the enzymatic activity detection device 16. According to example embodiments shown schematically in FIG. 15, the computing system 1500 can include a computing environment 1510, a user interface 1550, a communication unit 1560. The computing system can further include a haptic motor and an accelerometer. The computing environment 1510 can include a processor 1520, a memory 1530, and an I/O interface 1540. The computing environment 1510 can be coupled to the user interface 1550 and communication unit 1560 through the I/O interface 1540.

[00114] The processor 1520 can typically control the overall operations of the computing environment 1510, such as the operations associated with data acquisition, data processing, and data communications. The processor 1520 can include one or more processors to execute instructions to perform all or some of the steps in the above-described methods. Moreover, the processor 1520 can include one or more modules that facilitate the interaction between the processor 1520 and other components. The processor may be or include a central processing unit (CPU), a microprocessor, a single chip machine, a graphical processing unit (GPU) or the like.

[00115] The memory 1530 can store various types of data to support the operation of the computing environment 1510. Memory 1530 can include predetermined software 15 1. Examples of such data comprise instructions for any applications or methods operated on the computing environment 1510, raw data, detected data, etc. The memory 1530 may be implemented by using any type of volatile or non-volatile memory devices, or a combination thereof, such as a static random-access memory (SRAM), an electrically erasable programmable read-only memory (EEPROM), an erasable programmable read-only memory (EPROM), a programmable read-only memory (PROM), a read-only memory (ROM), a magnetic memory, a flash memory, a magnetic or optical disk.

[00116] The VO interface 1540 can provide an interface between the processor 1520 and peripheral interface modules, such as a RF circuitry, external port, proximity sensor, audio and speaker circuitry, video and camera circuitry, microphone, accelerometer, display controller, optical sensor controller, intensity sensor controller, other input controllers, keyboard, a click wheel, buttons, and the like. The buttons may include but are not limited to, a home button, a power button, and volume buttons.

[00117] The user interface 1550 can include a speaker, lights, display or other similar technologies for communicating with the user.

[00118] Communication unit 1560 provides communication between the processing unit, an external device, mobile device, and a webserver (or cloud). The communication can be done through, for example, WIFI or BLUETOOTH hardware and protocols. The communication unit 1560 can be within the computing environment or connected to it.

[00119] In some embodiments, there is also provided a non-transitory computer-readable storage medium comprising a plurality of programs, such as comprised in the memory 1530, executable by the processor 1520 in the computing environment 1510, for performing the above- described methods. For example, the non-transitory computer-readable storage medium may be a

ROM, a RAM, or the like.

[00120] The non-transitory computer-readable storage medium has stored therein a plurality of programs for execution by a computing device having one or more processors, where the plurality of programs when executed by the one or more processors, cause the computing device to perform the above-described method for motion prediction.

[00121] In some embodiments, the computing environment 1510 may be implemented with one or more application-specific integrated circuits (ASICs), digital signal processors (DSPs), digital signal processing devices (DSPDs), programmable logic devices (PLDs), field-programmable gate arrays (FPGAs), graphical processing units (GPUs), controllers, micro-controllers, microprocessors, or other electronic components, for performing the above methods.

[00122] From the foregoing, it will be observed that numerous modifications and variations can be effectuated without departing from the true spirit and scope of the novel concepts of the present disclosure. It is to be understood that no limitation with respect to the specific embodiments illustrated is intended or should be inferred. The disclosure is intended to cover by the appended claims all such modifications as fall within the scope of the claims.

Claims

CLAIMS What is claimed is:

1. A system for enzymatic activity detection comprising: a collecting unit, wherein the collecting unit collects dejecta entering an ostomy bag from a stoma; an enzymatic activity detection device, wherein the enzymatic activity detection device comprises an assay unit, wherein the assay unit comprises at least one assay chamber having an assay solution for detecting a target proteolytic enzyme; and an inlet valve, wherein the inlet valve is a one-way valve that transmits the dejecta from the collecting unit to the enzymatic activity detection device.

2. The system of claim 1, wherein the assay solution comprises a solution optimized for a fluorescence output based on a target enzyme concentration.

3. The system of claim 2, wherein the assay solution comprises a substrate that releases fluorescence upon cleavage.

4. The system of claim 2, wherein the assay solution comprises fluorophore and quencher-conjugated target proteins with specific cleavage motifs for detecting the target proteolytic enzyme.

5. The system of claim 1, wherein the assay unit further comprises a light source that provides light at an excitation wavelength to the solution.

6. The system of claim 1, wherein the assay unit further comprises a fluorometric sensor that measures fluorescent signals to detect a target proteolytic enzyme activity, released in response to an excitation light signal.

7. The system of claim 6, wherein the fluorometric sensor comprises a complementary metal-oxide semiconductor (CMOS) sensor.

8. The system of claim 1, wherein the at least one assay chamber comprises a first assay chamber configured to detect a first proteolytic enzyme activity and a second assay chamber configured to detect a second proteolytic enzyme activity, and wherein the first and second proteolytic enzymes are different.

9. The system of claim 1, wherein the inlet valve closes once a pressure gradient is met, and wherein the pressure gradient indicating a filling of the dejecta in the assay unit.

10. The system of claim 9, wherein the enzymatic activity detection device further comprises a waste unit for collecting dejecta that has passed through the assay unit.

11. The system of claim 1 , wherein the enzymatic activity detection device further comprises an analyzing unit that outputs detection information based on a presence of the target proteolytic enzyme.

12. A method for detecting enzymatic activity in ostomy dejecta to predict, mitigate or prevent peristomal skin complications (PSC)comprising: obtaining dejecta from a stoma; detecting, using an enzymatic activity detection device, at least one target proteolytic enzyme based on the dejecta and an assay solution; and outputting detection information based on the detected at least one target proteolytic enzyme, wherein the enzymatic activity detection device comprises an assay unit, the assay unit comprising at least one assay chamber having the assay solution, and an inlet valve, wherein the inlet valve is a one-way valve that transmits the dejecta from a collecting unit to the enzymatic activity detection device.

13. The method of claim 12, wherein the assay solution comprises a solution optimized for a fluorescence output based on a presence of the at least one target proteolytic enzyme.

14. The method of claim 13, wherein the assay solution comprises a red fluorescence ENZCHEK Protease assay.

15. The method of claim 13, wherein the assay solution comprises fluorophore and quencher-conjugated target proteins with specific cleavage motifs for detecting the at least one target proteolytic enzyme.

16. The method of claim 13, wherein detecting, using the enzymatic activity detection device, the at least one target proteolytic enzyme based on the dejecta and the assay solution comprises: measuring a fluorescent signal based on the dejecta and the assay solution; and detecting the at least one target proteolytic enzyme based on the fluorescent signal.

17. The method of claim 13, wherein detecting, using the enzymatic activity detection device, the at least one target proteolytic enzyme based on the dejecta and the assay solution comprises: detecting a first target proteolytic enzyme based on the dejecta and a first assay solution; and detecting a second target proteolytic enzyme based on the dejecta and a second assay solution, wherein the first and second target proteolytic enzymes are different.

18. The method of claim 13, further comprising: disposing of the dejecta in a waste unit.

19. The method of claim 13, wherein outputting detection information comprises: displaying the detection information, wherein the detection information comprises a potential PSC detection alert.