WO2018058077A1

WO2018058077A1 - System and method for urinary catheter bacterial detection

Info

Publication number: WO2018058077A1
Application number: PCT/US2017/053337
Authority: WO
Inventors: Victoria Y. BIRD; Brandey L. ANDERSEN; Kirk ZIEGLER; Anniruddah KULKARNI
Original assignee: University Of Florida Research Foundation, Inc.
Priority date: 2016-09-23
Filing date: 2017-09-25
Publication date: 2018-03-29

Abstract

Devices equipped with one or more sensors to detect pH changes or presence of nitrite in a fluid are disclosed herein. Specifically exemplified, are urinary catheter systems that are capable of detection pH and nitrite concentration changes in urine. Also disclosed are new hydrogel compositions that are capable of providing colorimetric changes in response to pH and nitrite concentration levels in a fluid to which the compositions are exposed.

Description

SYSTEM AND METHOD FOR URINARY CATHETER BACTERIAL DETECTION

BACKGROUND

[0001] Catheter-associated Urinary Tract Infections (CAUTI) are caused by bacterial growth in urinary catheters. CAUTI are the most common type of health care- associated infections, counting for over 30% of hospital associated infections. CAUTI are responsible for 13,000 deaths annually in the United States, increased hospital stays and an annual $0.5 Billion increase in the cost of healthcare. Recently, Medicare and private insurance companies ceased payment for Hospital-acquired CAUTI, as an incentive to prevent their instance. However, recent analysis has shown no statistically significant reduction in Hospital- acquired CAUTI. Therefore, there remains a need for the prevention of bacterial growth in urinary catheters.

BRIEF DESCRIPTION OF THE DRAWINGS

[0002] Embodiments are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings in which like reference numerals refer to similar elements and in which:

[0003] FIG. 1A is a schematic diagram that illustrates one example of a system for urinary catheter bacterial detection, according to an embodiment.

FIG. IB shows a urinary catheter system that incorporates a sensor coupling embodiment that includes one or more sensors to detect pH or nitrite.

FIG. 1C shows the sensor coupling of FIG. IB disassembled from the urinary catheter and collection tubing.

[0004] FIG. 2 shows a photograph of a urinary catheter tubing that includes a sensor that is disposed in the lumen thereof.

[0005] FIG. 3A shows a collection dish that include a plurality of sensors for detecting pH or nitrite, typically in a fluid specimen such as urine/

FIG. 3B shows a slide having a plurality of sensors disposed thereon.

[0006] FIG. 4 shows photographs of tubing that includes a pH sensor that have been subjected to fluids of varying pH/

[0007] FIG. 5 is embodiment graph showing a kinetic response time of pH sensors.

[0008] FIG. 6 is diagram illustrating the principle behind colorimetric changes in functionalized nanoparticles exposed to nitrite.

[0009] FIG. 7 is a graph showing the absorbance changes as a function of aggregation of nanoparticles.

[0010] FIG. 8A represent TEM image of dispersed nanoparticles.

FIG. 8B shows aggregated nanoparticles. FIG. 8C shows dispersed nanoparticles that disaggregate in response to nitrite.

[0011] FIG. 9 is a photograph solutions of gold nanoparticles that have been subjected to varying concentrations of nitrite.

[0012] FIG. 10 is an absorbance spectra graph showing the absorbance response of gold nanoparticles to varying concentrations of nitrite

[0013] FIG. 11. is a series of photographs showing hydrogel compositions containing functionalized gold nanoparticles embedded therein.

[0014] FIG. 12 is a photograph showing the colorimetric change of the nanoparticle containing hydrogel composition when exposed to nitrite.

DETAILED DESCRIPTION

[0015] Disclosed herein are urinary catheter systems that are equipped with unique sensors configured to reversibly detect pH changes over prolonged periods of time. Accordingly to one embodiment, disclosed is a sensor coupling for connecting a urinary catheter and tubing of a urine collecting bag. The sensor coupling includes a hollow body that defines a channel for passing fluid, the body comprising a first end for connecting with the urinary catheter and a second end for connecting to the tubing and at least one sensor associated with the sensor coupling for detecting a bacterial presence in urine flowing through the channel. The at least one sensor includes a pH sensor that reversibly provides a colorimetric change based on a pH level in the urine, or a nitrite concentration sensor that provides a colorimetric change based on a nitrite concentration in the urine or a combination of the pH sensor and the nitrite concentration sensor.

[0016] The pH sensor may be comprised of a hydrogel and a dye encapsulated by the hydrogel, wherein the dye provides a colorimetric change over a physiological pH range of the urine. In a specific embodiment, the dye is bromothymol blue dye (BTB) and the physiological pH range is approximately 6-8. In a more specific embodiment, the pH sensor detects a change in urine pH from below a pH of 6-8 to a pH of 6-8 or above. Typically, the response time between exposure of the dye to urine at the pH level and the first distinct visual color change to the pH level is in a range of 3-100 seconds.

[0017] When the sensor is a nitrite sensor, the sensor is comprised nanoparticles having plasmonic properties embedded in a hydrogel matrix. In a specific embodiment, the nanoparticles may include gold nanoparticles or copper nanoparticles. Typically, the nanoparticles are functionalized with a disulfide ligand associated with an amine. The amine typically will be one that binds strongly to the nanoparticles but not too long or large so as to interfere with the plasmon resonance of the nanoparticles. In a specific embodiment, the amine is an aromatic amine. The term "functionalized" as used herein means the

nanoparticles are treated such that a reactive compound is associated with the surface of the nanoparticle. A disulfide ligand-amine complex causes inducement of aggregation of nanoparticles which is reactive with nitrite to disaggregate the nanoparticles. The aggregated and disaggregated nanoparticles have different light absorbance and produce visually detectable differing colors. In an even more specific embodiment, the functionalized nanoparticles are gold particles coated with 4-aminothiol disulfide.

[0018] Another embodiment pertains to urinary catheter system comprising a conduit in fluid communication with a urine collection bag so as to allow urine from a subject to flow through the conduit to the urine collection bag, the system further comprising a reversible pH sensor or a nitrite sensor, or both, disposed within the system so as to be exposed to urine flowing in the conduit or urine collection bag. The pH sensor provides a colorimetric change in response to a pH change from below 6-8 to 6-8 or above and is capable of such

colorimetric change after 24 hrs, 48 hrs, 72 hrs, 168 hrs, 240 hrs or more exposure to urine; and the nitrite sensor comprises nanoparticles having plasmonic properties embedded in a hydrogel such that a threshold level of nitrite in the urine causes disaggregation of the nanoparticles that induces a colorimetric change.

[0019] According to a further embodiment, disclosed is a method for monitoring for urine infections indicative of an infection in a subject catheterized with the urinary catheter system. The method involves exposing a pH sensor of the system to urine for at least 24-240 hrs, and detecting a colorimetric change from a first color to a second color in the pH sensor responsive to a change in urine pH from below 6-8 to 6-8 or above. Moreover, upon detection of the first colorimetric change, the method further involves monitoring for a reversal of the colorimetric change from the second color back to the first color after a predetermined period of time or after treating the patient for an infection, or both. Reversal of the pH sensor is indicative of an alleviation of any potential infection. The system may further include a nitrite sensor, and upon exposure of the nitrite sensor to urine for at least 24- 240 hrs, the method further involves detecting a colorimetric change in the nitrite sensor. The system may include both a pH sensor and nitrite sensor, and if both undergo a colorimetric change, the method further comprises replacing the urinary catheter system. Alternatively, upon colorimetric changes in both the pH sensor and the nitrite sensor, the subject is treated for an infection and the pH sensor is monitored for a period of time. If the pH sensor reverses color, this indicates that the treatment of infection is successful.

[0020] A further embodiment pertains to a method for making a pH sensing membrane, wherein method involves

a) dissolving polymer in a solvent to form a solution;

b) mixing an amount of at least one indicator dye in the solution; c) mixing a lipophilic salt in the solution of step a) or mixture of step b);

d) adding a plasticizer to the mixture of step b) or c); and

e) casting an amount of the mixture of step d) onto a surface.

The term "membrane" as used here and throughout the description refers to a type of sensor in the form of one or more layers of a composition disposed on a surface of an object. In a specific embodiment, the polymer may include but is not limited to one of polyvinyl chloride, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene oxide, polyhydroxyethyl methacrylate, alginate, or cellulose acetate, or a combination thereof. In another specific embodiment, the indicator dye may include but is not limited to one of malachite green; methyl violet 10B; thymol blue; methyl yellow; bromophenol blue; congo red; methyl orange; bromocresol green; methyl red; bromocresol purple; bromothymol blue; phenol red; neutral red; naphtholphthalein; cresol red; phenolphthalein; thymolphthalein; alizarine yellow R; or Indigo Carmine; or combinations of the foregoing. In a more specific embodiment, the at least one indicator dye may include an amount of bromothymol blue and an amount of methyl red and/or cresol red.

[0021] The solvent utilized in the method for making a pH sensing membrane described above may include, but is not limited to, one of tetrhydrofuran; touluene; benzene; 1,1,1 Trichloroethane; xylene; carbon tetrachloride; ethyl acetate; chloroform; trichloroethylene; cellosolve® acetate; methyl ethyl ketone; acetone; diacetone alcohol; ethylene dichloride; methylene chloride; butyl cellosolve®; water; glycerol; or ethylene glycol; or a combination thereof.

[0022] The lipophilic salt utilized in the method for making a pH sensing membrane described above may include, but is not limited to, one of Aliquat 336;

dimethyldioctadecylammonium bromide (DDA); tridodecylmethylammonium chloride (TDMAC); or potassium tetrakis(4-chlorophenyl borate (TCPB), or a combination thereof.

[0023] The plasticizer utilized in the method for making a pH sensing membrane described above may include, but is not limited to, one of O-nitrophenyloctylether (NPOE); dioctyl sebacate (DOS); bis(l-butylpentyl)adipate (BBPA); or tributyl phosphate (TBP); or a combination thereof.

[0024] According to other embodiments, provided is an article of manufacture including a pH sensor disposed on a surface thereon, wherein the pH sensor comprises a composition comprising an indicator dye embedded in a hydrogel and the article comprises a catheter, a dipstick, a funnel, a dish, a container, a diaper, or an absorbent pad.

[0025] A more specific synthesis method for making a pH sensing membrane involves the following:

a) dissolving polyvinyl chloride in a solvent to form a polymer solution; b) mixing an amount of bromothymol blue in the polymer solution;

c) mixing an amount of Aliquat 336 to the mixture of step b); d) adding NPOE to the mixture of step c); and e) casting an amount of the mixture of step d) onto a surface.

[0026] Another embodiment disclosed herein pertains to a method of detecting the presence of nitrite in a liquid sample, the method involves

obtaining a population of aggregated gold nanoparticles comprising disulfide ligands bound to the surface thereof, wherein the population of aggregated gold nanoparticles possess a first color ; and

subjecting the population to an amount of the liquid sample, wherein a presence of nitrite in the liquid sample causes disaggregation of the gold nanoparticles that generates a color change from the first color to a second color. In one example, the aggregated gold nanoparticles are in a liquid suspension or solution and the liquid sample is added to such suspension or solution. In another example, the aggregated gold nanoparticles are fixated in a porous hydrogel.

[0027] Also disclosed herein is a porous hydrogel composition comprising a population of aggregated gold nanoparticles with disulfide ligands bound to the surface thereof. Also dislosed are articles of manufacture having a membrane of such hydrogel composition. The article may include, but not limited to, at least one of a catheter, a dipstick, a funnel, a dish, a container, a diaper, an absorbent pad, or a catheter coupling device.

[0028] Another method of making a composition colorimetrically responsive to nitrite involves:

a) Dissolving a polymer in a solvent to form a polymer solution; b) Dissolving sulfanilic acid in a water containing solvent to form an sulfanilic solution,

c) mixing the solutions of a) and b) with an amount of plasticizer; and d) casting an amount of the mixture of c) onto a surface. Another embodiment pertains to a membrane formed by this method.

[0029] A further embodiment pertains to a method of making a composition

colorimetrically responsive to nitrite. This method involves a) Dissolving a polymer in a solvent to form a polymer solution;

b) mixing an amount of gold nanoparticles in the polymer solution, and c) casting an amount of the mixture of b) onto a surface. The method may further involve subjecting the gold nanoparticles to a functionalization step to associate a disulfide ligand to the surface of the nanoparticles, wherein the functionalization step occurs prior to step b), prior to step c) or after step c).

Overview

[0030] Bacterial infection of urinary catheters is caused by urease-producing organisms and nitrite producing organisms, such as Proteus mirabilis, Pseudomonas aeruginosa and Escherichia coli, among others, that colonize the catheter and form a biofilm. The bacterial urease generates ammonia from urea and the urine becomes alkaline. Under these conditions, crystals of calcium and magnesium phosphate are formed and a crystalline biofilm develops, which eventually blocks the flow of urine from the bladder. pH of urine can be used as an indicator that the urine is becoming alkaline (typically as a result of infection) and thus can be used to prevent blockage of urinary catheters. In addition, nitrites which are made by some bacteria that commonly colonize urethral catheters and the urinary tract is also used worldwide to detect urinary tract infections (UTI's). Nitrite detection is a powerful tool used to detect UTI since humans do not have the ability to make nitrites in urine. Currently, the only way to detect CAUTI in hospitalized patients is through symptoms of fevers and increase temperature in bladder and body temperature, which happens when bacteria already has caused marked inflammation, infection and possible sepsis. By detecting nitrite levels in urine with high sensitivity, it may be useful in determining possible bacteria within bladder before it has caused infection and consequently prevent sepsis and determine the need for intervention either through antibiotics and/or replacement of the catheter. Provided herein are systems and methods of use thereof for determining characteristics of urine in a catheterized patient that will assist health-care providers in addressing such medical issues. [0031] Conventional pH sensors have been developed, such as a cathechol conjugated alginate hydrogel encapsulated with pyrocatechol violet dye¹. However, it has been recognized that the pH sensor disclosed in Lee has notable drawbacks. For example, the pH sensor in Lee provides weak color change in a wide pH range (e.g. 2-7) and thus is only useful for detecting strong acid or strong base pH levels. Accordingly, it is further recognized that the pH sensor in Lee would not be useful for providing noticeable color change across a physiological range (e.g. 6-8) for bacterial detection in a urinary catheter. The present application is based on the development of a pH sensor that produces noticeable color change across the physiological range (e.g. 6-8) to visually detect when the pH reaches an elevated level (e.g. 7.5) indicating that the catheter needs to be changed.

Detailed Description of Illustrated Embodiments

[0032] Certain embodiments disclosed herein include a pH sensor that is reversible and is able to indicate fluctuations in urine pH over time. In one embodiment, the system described herein is provided to detect changes in urine pH and prevent CAUTI by acting immediately and changing the catheter with sterile techniques. In some embodiments, the role of the system is short term hospital stays either on the medical or surgical inpatient floors or MICU (Medical Intensive Care Unit) or SICU (Surgical Intensive Care Unit).

[0033] FIG. 1A is a schematic diagram that illustrates one example of a system 10 for urinary catheter bacterial detection, according to an embodiment. The system 10 includes a urine collecting bag 12 and associated collecting tubing 14 that is connected at a sensor coupling 15 to a urinary catheter 16. As appreciated by one of ordinary skill in the art, the catheter 16 is inserted into the bladder through the urethra (not shown).

[0034] FIGS. 1B-1C illustrate the sensor coupling 15 between the urine catheter 16 and urine collection bag 12 in the system 10 of FIG. 1A, according to an embodiment. The sensor coupling 15 includes a hollow body with a channel 15C for passage of urine through the coupling and into the collecting tubing 14. One or more colorimetric sensors 17a-f designed for producing colorimetric changes in response to pH change or nitrite

Yu-Kyoung Lee, A colorimetric alginate-catechol hydrogel suitable as a spreadable pH indicator, Dyes and concentration are associated with the sensor coupling and positioned so as to contact urine passing through the channel 15C.

[0035] In some embodiments, the sensor coupling 15 is typically made of clear material and the one or more sensors 17 are positioned in the channel whereby a user can visually observe a change in color of the sensor 17. In an example embodiment, the clear material is one of Silicon, polyurethane, latex, mixed plastic polymer, or polyvinyl chloride (PVC). However, the material of the sensor coupling 15 is not limited to these listed materials. The sensor coupling 15 includes a body with two ends, with a first end 19 configured to connect with the urinary catheter 16 and a second end 21 opposite to the first end that is configured to connect to the tubing 14 of the urine collecting bag. The first end 19 features a tapered tip 26 that is fixedly inserted in an end of the catheter 16. The tapered tip 26 may be ribbed and shaped such that it fits in the end of a variety of sized catheters 16.

[0036] In specific example, an outer diameter of the sensor coupling 15 is in a range of 8-15 mm. In another example embodiment, an inner diameter of the sensor coupling 15 is in a range of 7-12 mm. In some embodiments, the sensor coupling is configured as a universal device to fit into any sized tubing related to catheters or drainage bags, including suprapubic and nephrostomy tubing or any other tubing related to the urinary tract. The sensor coupling preserves a sterility of the system 10 and is disposable once catheter 16

replacement occurs.

[0037] Although the embodiment of FIG. 1 depicts that the sensor coupling features a step- down tip 26, the embodiments of sensor coupling are not limited to this arrangement. The sensor coupling is not limited to the numerical dimensions and can simply be a tapered tip that slides into a tubing. The sensor coupling is typically made of transparent material where the one or more sensors are disposed.

[0038] FIG. 2 shows an embodiment pertaining to a tube 40 having a pH sensor disposed on an inner lumen thereof. The sensor includes bromo-thymol blue embedded in a hydrogel matrix. Upon exposure to an elevated pH, the sensor turns from yellow to green. It will be

Pigments 108 (2014) 1-6 ("Lee") understood that sensors described herein can be implemented in a variety of different devices. Such devices include a catheter, a dipstick, a funnel, a dish, a container, a diaper, or an absorbent pad. The sensors can be exposed to a biological sample such as urine and the change in pH or presence of nitrite, as described further herein, can be detected. In a specific embodiment, elevated pH and presence of nitrite is used to diagnose urinary tract infections.

[0039] FIG. 3A shows one alternative embodiment of a medical device (a collection dish 50) that includes one or more sensors 17 for detection of pH changes or presence of nitrite. FIG. 3B shows another alternative embodiment (a slide 52) that also includes one or more sensors 17disposed thereon.

[0040] In some embodiments, the one or more sensors 17 is a pH sensor that provides a distinct visual color change over a physiological pH range (e.g. 6-8). The pH sensor provides a distinct visual color change at an elevated pH level (e.g. 7.5) indicating that the catheter 16 requires replacement. The pH sensor also provides reversible color change over the physiological pH range and thus responds to changes in pH over time. In one embodiment, the sensor 17 is a hydrogel that encapsulates a color changing dye over the physiological pH range. In one example embodiment, the sensor is a hydrogel that has bromothymol blue dye (BTB) embedded therein, which provides a distinct visual color change from yellow to blue over the physiological pH range. Indeed, BTB is merely one example embodiment of a color changing dye encapsulated by the hydrogel. Other types of color changing dyes can be developed and/or used, and similarly encapsulated by the hydrogel, provided that they similarly exhibit a visual color change over the physiological pH range. The Examples Section below sets forth a nonexclusive list of other dyes that can be used to exhibit visual color change for pH ranges outside of the physiological range.

[0041] In some embodiments, a time of color change of the pH sensor, measured as a time delay between exposure of the pH sensor to urine at a pH level and a distinct color change of the pH sensor to visually indicate the pH level, is in a range of 1-100 seconds. In some embodiments, the pH sensor is configured to have a pH triggered change within the above time window and remain at that color as long the pH level of the urine remains stable. The H sensor advantageously provides reversible change to avoid misinterpretation when a patient receives an alkali load, such as bicarbonate, that transiently rises the pH level.

[0042] In other embodiments, the sensor 17 is a nitrite concentration sensor that provides a distinct visual color change based on a change in nitrite concentration of urine flowing through the sensor coupling 15. The presence of nitrite ions in the urine is a definitive indication of bacterial colonization in the urinary tract. The nitrite concentration sensor provides a colorimetric assay with visual, on-site analysis with a simple and instantaneous detection method. In some embodiments, the nitrite concentration sensor involves colorimetric detection of nitrites by interacting the nitrites with amine coated nanoparticles having plasmonic properties.

[0043] In an example embodiment, the nitrite concentration sensor includes a colorimetric assay based on anti-aggregation of nanoparticles (e.g. Au nanoparticles). In a specific example, gold nanoparticles coated with aromatic amines react with nitrite ions in the urine which alters the aggregation state of the nanoparticles. This change in the aggregation state of the nanoparticles causes a shift in the local surface plasmon resonance bands, triggering a visual colorimetric response. In some embodiments, the change in the aggregation state of the nanoparticles also causes the formation of diazoium salt. The level of change in the aggregation state and resulting level of colorimetric response, is dependent on the concentration of nitrite ions in the urine. In an example embodiment, the nitrite concentration sensor provides a distinct visual color change over a range of nitrite concentration, such as from red to purple to blue. However, the distinct visual color change is not limited to any particular range of colors and includes all ranges of visually distinct color changes over the range of nitrite concentration. In one example embodiment, thiol-based linkers are used to aggregate the gold nanoparticles in the nitrite concentration sensor. In another example embodiment, disulfide cross-linkers, such as 4-aminothiol disulfide, is used to aggregate the gold nanoparticles in the nitrite concentration sensor.

[0044] The amine coated nanoparticles discussed above are merely one example embodiment of the nitrite concentration sensor. Other sensors or techniques can be developed and/or used for the nitrite concentration sensor 17. In an example embodiment, another sensor or technique includes covalent/co-crosslinked attachment of azo dye to PEG- based hydrogel. Further information on compositions suitable for use in nitrite detection and methods of making the same are provided in the Examples Section.

[0045] It is contemplated that more than one sensor 17 may be utilized in the catheter tubing, collection tubing, collection bag or sensor coupling 15, or the other implementations discussed above. For example, the one or more sensors 17 may be two or more different pH sensors adapted to detect pH at differing ranges. Typically, this would involve a first pH sensor that include a first dye embedded in a gel matrix and a second pH sensor that includes a second dye embedded in a gel matrix. In addition, the one or more sensors 17 can include a nitrite sensor that detects the presence of nitrite, as is further described herein. As described below, alterations in the synthesis protocol of the nitrite sensor can affect the color or other properties of the sensor. Therefore, there can be nitrite sensors that utilize nanoparticles and nitrite sensors that utilize a Griess reagent. Accordingly, the one or more sensors 17 can include a pH sensor and a nitrite sensor, or a plurality of pH sensors, a plurality of pH sensors and a nitrite sensor, a pH sensor and a plurality of nitrite sensors, a plurality of nitrite sensors, or a plurality of both pH sensors and nitrite sensors.

Examples

Example 1: Durable and Fluid Resistant pH or Nitrite Sensors

[0046] Provided below are examples of compositions useful for implementation with various devices for detecting pH changes in a fluid such as urine and protocols for synthesizing the same. The protocols are utilized for electrostatic retention of dye or detection agent within polymer. There can be many permutations and slight variations within these protocols. For example, rather than using sulfuric acid in the synthesis of the nitrite- detection membrane, one could use another acid. Additionally, the volumes of the

corresponding entities can also be modified. These measures and additional improvements can be made in order to ensure synthesis of a robust membrane with ideal colorimetric properties and little to no leaching. Also, solvents, dyes, polymers, plasticizers and lipophilic salts of the general protocol can be substituted as set forth below. Synthesis Protocol for pH-Sensitive Membrane

I . Obtain 20 mL scintillation vial and clean with tetrahydrofuran (THF) and allow to dry

2. Add 5 mL of THF to the vial using a positive-displacement pipette

3. Weigh out 274 mg of high Molecular Weight Polyvinylchloride

4. Slowly add PVC to vial containing THF*

5. Vortex and shake/incubate the resultant solution at 37°C and 130 rpm overnight

6. Add 15mg of indicator dye to the solvated PVC solution. If more than one is present, then 7.5 mg each, etc.

7. Vortex for 1-3 minutes

8. Add 57 μL· of the lipophilic salt, Aliquat 336, to the solution

9. Vortex for 1-3 minutes

10. Add 635 μΐ_^ of plasticizer, NPOE, to the solution

I I. Vortex for 1-3 minutes

12. Cast membrane on material and wait 30 minutes for membrane to dry

13. Test membrane with pH solution

Perform synthesis in hood, since THF should not be inhaled

Synthesis Protocol for Nitrite-Sensitive Membrane:

1. Obtain 20 mL scintillation vial and clean with tetrahydrofuran (THF) and allow to dry

2. Add 5 mL of THF to the vial using a positive-displacement pipette

3. Weigh out 274 mg of high Molecular Weight Polyvinylchloride

4. Slowly add PVC to vial containing THF*

6. Boil 5 mL of water and dissolve 13.7 mg sulfanilic acid it in the boiled water 7. Add 100 mL of 2.0 N H2S04 into the resultant solution to aid in dissolution

8. Vortex for 4-5 minutes or until mixture is homogenous

9. Add 635 μΐ_^ of plasticizer, NPOE, to the solution

10. Vortex for 1-3 minutes

11. Cast membrane on material and wait 30 minutes for membrane to dry

12. Test membrane with nitrite solution

* Perform synthesis in hood, since THF should not be inhaled.

Indicator Dye Options

[0047] Various indicator dyes may be included in the membrane matrix to allow for detection of the pH of the surrounding aqueous environment. Below are several examples of dyes that are relevant to the physiology of the urinary environment.

Malachite Green

- Yellow-Green, pH 0.0-2.0

- Green-Translucent, pH 11.6-14.0

Gentian Violet (Methyl Violet 10B)

- Yellow-Blue/Violet, pH 0.0-2.0

Thymol Blue

- Red- Yellow, pH 1.2-2.8

- Yellow-Blue, pH 8.0-9.6

Methyl Yellow

- Red- Yellow, pH 2.9-4.0

Bromophenol Blue

- Yellow-Blue, pH 3.0-4.6 Congo Red

- Blue/Violet-Red, pH 3.0-5.0

Methyl Orange

- Red-Gray, pH 0.0-3.2

- Gray-Green, pH 3.2-4.2

Bromocresol Green

- Yellow-Blue, pH 3.8-5.4

Methyl Red

- Red- Yellow, pH 4.4-6.2

Bromocresol Purple

- Yellow-Purple, pH 5.2-6.8

Bromothymol Blue

- Yellow-Blue, pH 6.0-7.6

Phenol Red

- Yellow-Red, pH 6.4-8.0 Neutral Red

- Red- Yellow, pH 6.8-8.0

Naphtholphthalein

- Pale Red-Green/Blue, pH 7.3-8.7

Cresol Red

- Yellow-Red/Purple, pH 7.2-8.8 o-Cresolphthalein

- Colorless-Purple, pH 8.2-9.8

Phenolphthalein

- Colorless-Purple/Pink, pH 8.3-10.0

Thymolphthalein

- Colorless-Blue, pH 9.3-10.5

Alizarine Yellow R

- Yellow-Red, pH 10.2-12.0

Indigo Carmine

- Blue- Yellow, pH 11.4-13.0

[0048] The membrane is typically hydrophilic and can be a synthetic membrane, such as a polyvinyl chloride, or natural membrane, such as a cellulose or alginate. The membrane could also be positively, negatively, or neutrally charged. Below are a few examples of possible composition:

Polymers

[0049] The membranes can be naturally and or synthetically fabricated using liquids, metals, ceramics, biomaterials, heterogeneous solids, homogeneous films, inorganic materials, and organic materials. Any such membrane that is sensitive to pH and nitrite detection. Derivatives of these polymers are included as well. Examples include but are not limited to polyvinyl chloride; polyethylene glycol; poly(N-2-hydroxypropyl

methacrylamide); poly(N-isopropylacrylamide); polylactic acid; polyhdryoxyalkanoates; polycaprolactone; poly(propylene fumarate); polyanhyrdrides; polyacetals; poly(ortho esters); polycarbonates; polyurethanes; polyphosphoesters; polyphosphazenes; alginate (e.g. catehchol conjugated alginate hydrogel); cellulose acetate; synthetic polymer derivatives; and combinations of the foregoing. Other compositions useful for making sensors include liquid emulsions; polymer-electrolytes; and ceramics.

Lipophilic Salt

[0050] Any quaternary ammonium lipophilic salt and its derivatives is considered for the synthesis of the membrane. This includes quaternary lipophilic countercations and lipophilic counteranions. Additionally, the use of hydrophilic salts, hydrophobic salts, cationic salts, anionic salts, and ionic salts is possible. Examples include but are not limited to Aliquat 336; dimethyldioctadecylammonium bromide (DDA); Tridodecylmethylammonium chloride (TDMAC); and Potassium tetrakis (4-chlorophenyl) borate (TCPB).

Solvent

[0051] Solvents that have a similar Hildebrand solubility parameter to that of the solute (the polymer), are considered in the synthesis of the overall membrane. Examples include but are not limited to Tetrahydrofuran THF; Toluene; Benzene; 1,1,1 Trichloroethane;

Xylene; Carbon Tetrachloride; Ethyl acetate; Chloroform; Trichloroethylene; Cellosolve® acetate; Methyl ethyl ketone; Acetone; Diacetone alcohol; Ethylene dichloride; Methylene chloride; Butyl Cellosolve® ; Water; Gylcerol and Ethylene glycol.

Plasticizer

[0052] Additional plasticizers may also be used:

O-nitrophenyloctylether NPOE

Dioctyl sebacate DOS

bis(l-butylpentyl)adipate BBPA

Tributyl phosphate TBP

[0053] Further, once the membrane is formulated it can be cast on a variety of surfaces of a number of clinical, diagnostic, and biofeedback applications. Some of these include within catheters, dipstick formulations, funnels, weigh boats, and a number of plastics. pH Sensitivity in Catheter Tubing

[0054] FIG. 4 demonstrates the pH sensitivity of a gel composition embodiment made according to the protocols outlined above as well as the reliable reversibility of the colorimetric change of the composition. FIG. 4 shows the responsiveness of a pH sensor embodiment that can be implemented int a number of medical devices. As shown in FIG. 4, the sensor is coated on the inside of medical tubing. The indicators are all yellow before being subjected to any solutions. FIG. 4B shows that the colorimetric response of the indicator when subjected to solutions of varying pH. As shown, the color of the indicator changes from yellow to green when subjected to a solution at pH 6 or higher. Most important, all of the indicators demonstrate a reversal to the original yellow color when subjected to a low pH solution (see FIG. 4C).

[0055] Table 1 below lists specific examples typically used in synthesis of the membrane sensors.

Table 1

polyvinyl chloride (PVC) Polymer for Avg MW = 233,000 formation of plastic membrane

tetrahydrofuran (THF) Solvent

bromothymol blue (BTB) pH indicator dye for pH range 6.0 - 7.6

Thymol blue pH indicator dye for pH range 8.0 - 9.6

Example 2: Nanoparticle assay and Gel Compositions for Nitrite Detection

[0056] Nanoparticles can be surface-functionalized with various ligands (Elghanian et al. 1997; Storhoff et al. 1998). These ligands can control the size and dispersibility of the nanoparticles (Kim et al. 2004). The color (absorbance) of these solutions are strongly dependent on these changes to size and dispersion. As shown in FIG. 7, the aggregation of gold nanoparticles alters the distance-dependent surface plasmon resonance, resulting in a shift in the visible absorption spectrum. These shifts in absorbance are quite dramatic, allowing the colorimetric response to be observed by the naked eye. By designing the ligands to be reactive to a specific analyte (e.g., metabolite or biomarker), the dispersion of gold nanoparticles can be altered. FIG. 6 provides a diagram showing one scheme that has been devised for detection of nitrite based on an alteration of surface plasmon resonance due to aggregation and anti-aggregation of the nanoparticles. The gold nanoparticles in FIG. 6 have been functionalized with a disulfide ligand. The ligands on the gold surface act as a cross-linking agent between nanoparticles that causes them to aggregate into small clusters (blue color). These ligands react with nitrite ions and disrupt this cross-linking mechanism between nanoparticles. As a result of this reaction, the nanoparticles become dispersed again (purplish-red color). FIG. 8A-C provide TEM images showing the aggregation process of the nanoparticles.

Preparation of Gold Nanoparticles (AuNPs)

[0057] For every Au nanoparticle synthesis reaction thereafter, the beakers were rinsed with soapy water, ethanol and deionized water 3 times and dried in an oven at 100 °C before use. A stock solution of 1% Na3-citrate was prepared by addition of 1 g to 100 mL DI water. In a typical synthesis experiment, 0.1 g of HAuC14 was dissolved in 50 mL DI water in a beaker that was then put on a hot plate at 215 °C until the solution boiled. Once the solution began fuming and bubbles formed at the base of the beaker (typically -15 minutes), 2 mL of 1% Na3-citrate was added to the reaction mixture. The initial faint yellow color of the solution became colorless quickly and then rapidly became dark (in ~1 min). This dark color changed to purple red and finally wine red in about 10 minutes. The stirring rate was kept at -800 rpm throughout the experiment. This solution was allowed to cool for 30 minutes while stirring. For functionalization, the citrate stabilized gold nanoparticles are exposed to disulfide under conditions similar to that below for the nitrite detection but altered to substitute in the disulfide for association with the nanoparticles. Nitrite detection experiment

• ΙΟμΙ. of 0.1M H2S04 was added to 50μΙ. of 25μΜ 4- ATP and shaken for about a minute.

• To the above solution, 40μΙ. of 20mM NaN02 was added. The resultant solution mixture was kept aside for 15 minutes.

• 0.4ml of pH 10.01 buffer was added followed by the addition of 2mL of gold nanoparticles.

• Instant colour change was observed from red to blue.

[0058] While others have used 4-aminothiophenol ligands on gold nanoparticles to detect nitrite (Ye et al. 2015), we have synthesized gold nanoparticles with a disulfide ligand that posseses a strong affinity for the gold surface. The disulfide ligand is anticipated to form a stronger binding to the gold surface than common thiol-based ligands. The protocol for functionalizing the gold nanoparticles is similar to the nitrite detection above. Although the literature has suggested that disulfide ligands decompose into two thiol-based ligands upon adsorption to gold, the results produced herein suggest that the chemical functionality of these ligands is unique. For example, experiments have been conducted with both the disulfide and analogous thiol-based ligand and found that the sensitivity to nitrite is several orders of magnitude higher.

[0059] These nanoparticle-based sensors (in 2.5 mL of solution) are able to detect nitrite at concentrations of ~8 nM, which is 1500 times more sensitive than current colorimetric sensors (Ye et al. 2015). FIG. 9 shows the results of an experiment to observe the color change of the nanoparticle solutions at various concentrations of nitrite. FIG. 9 shows a change between 160nM and 1.6 μΜ concentrations that clearly visible by the naked eye. The detection range of nitrite concentration can be adjusted by altering the amount of ligand implemented. FIG. 10 pertains to an absorbance spectra that shows that the detection limite of the sensors is just above 6.4 nM.

Nanoparticle Hydrogels for Nitrite Detection Sensors

[0060] FIG. 11 shows hydrogel compositions that include the functionalized gold nanoparticles described above. FIG. 11 shows that the order in which the nanoparticles are added to the hydrogel affects the initial color of the hydrogel. Most importantly, FIG. 12 shows that hydrogel compositions including the functionalized gold nanoparticles allow for responsiveness to nitrite and result in a colorimetric change even while embedded in a hydrogel matrix. This discovery provides for the ability to implement hydrogel sensors onto a substrate such that nitrite can be detected. Gels containing the functionalized gold nanoparticles include those made from biocompatible polymers, such as the polymers listed in Example 1 above. For the hydrogel synthesis, the citrate stabilized gold nanoparticles are mixed with disulfide and the mixture is added to the hydrogel formulation.

[0061] Also, in view of the teachings herein, those skilled in the art will recognize that other metal nanoparticles having similar surface plasmonic properties can be substituted for the gold nanoparticles. One specific example of such metal nanoparticle includes copper nanoparticles.

[0062] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word "comprise" and its variations, such as "comprises" and "comprising," will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps. Furthermore, the indefinite article "a" or "an" is meant to indicate one or more of the item, element or step modified by the article. As used herein, unless otherwise clear from the context, a value is "about" another value if it is within a factor of two (twice or half) of the other value. While example ranges are given, unless otherwise clear from the context, any contained ranges are also intended in various embodiments. Thus, a range from 0 to 10 includes the range 1 to 4 in some embodiments. The teachings of any cited references are incorporated herein in their entirety to the extent they are not inconsistent with the teachings herein.

Claims

CLAIMS:

1. A sensor coupling for connecting a urinary catheter and tubing of a urine collecting bag, said sensor coupling comprising:

a hollow body that defines a channel for passing fluid, the body comprising a first end for connecting with the urinary catheter and a second end for connecting to the tubing; at least one sensor associated with the sensor coupling for detecting a bacterial presence in urine flowing through the channel;

wherein said at least one sensor comprises a pH sensor that reversibly provides a

colorimetric change based on a pH level in the urine, or a nitrite concentration sensor that provides a colorimetric change based on a nitrite concentration in the urine or a combination of the pH sensor and the nitrite concentration sensor.

2. The sensor coupling of claim 1, wherein said pH sensor includes a hydrogel and a dye encapsulated by the hydrogel, wherein the dye provides a colorimetric change over a physiological pH range of the urine.

3. The sensor coupling of claim 2, wherein the hydrogel is a catechol conjugated alginate hydrogel.

4. The sensor coupling of claim 2 wherein the dye is bromothymol blue dye (BTB).

5. The device of claim 2, wherein the physiological pH range is approximately 6-8.

6. The device of claim 5, wherein the pH sensor detects a change in urine pH from below a pH of 6-8 to a pH of 6-8 or above.

7. The device of claim 2, wherein a response time between exposure of the dye to urine at the pH level and the first distinct visual color change to the pH level is in a range of 3-100 seconds.

8. The device of claim 1, wherein the nitrite concentration sensor comprises nanoparticles having plasmonic properties.

9. The device of claim 8, wherein the nanoparticles are gold nanoparticles or copper nanoparticles.

10. The sensor coupling of claim 9, wherein the nanoparticles are functionalized with an amine associated with or without a disulfide ligand.

11. The sensor coupling of claim 10, wherein the amine is an aromatic amine.

12. The device of claim 11, wherein the amine coated nanoparticles are gold particles coated with 4-aminothiol disulfide.

13. The sensor coupling of claim 1, wherein the first end is a ribbed tapered tip that is configured to be fixedly inserted into an opening of the urinary catheter and wherein the second end is an opening that is configured to receive a tapered tip of the tubing of the urine collecting bag.

14. A urinary catheter system comprising a conduit in fluid communication with a urine collection bag so as to allow urine from a subject to flow through the conduit to the urine collection bag, the system further comprising a reversible pH sensor or a nitrite sensor, or both, disposed within the system so as to be exposed to urine flowing in the conduit or urine collection bag; wherein the pH sensor provides a colorimetric change in response to a pH change from below 6-8 to 6-8 or above and is capable of such colorimetric change after 24 hrs, 48 hrs, 72 hrs, 168 hrs, 240 hrs or more exposure to urine; and wherein the nitrite sensor comprises nanoparticles having plasmonic properties embedded in a hydrogel such that a threshold level of nitrite in the urine causes disaggregation of the nanoparticles that induces a colorimetric change.

15. The system of claim 14, wherein the nanoparticles are functionalized with a disulfide ligand.

16. The system of claim 15, wherein the nanoparticles functionalized with a disulfide ligand are gold nanoparticles coated with aromatic amines bound to the disulfide ligand.

17. The system of claim 15, wherein the nanoparticles are gold particles coated with 4- aminothiol disulfide.

18. The system of claim 14, wherein the pH sensor comprises a dye embedded in a hydrogel matrix.

19. The system of claim 18, wherein the dye is BTB and the hydrogel matrix is catehchol conjugated alginate.

20. A method for monitoring for urine infections indicative of an infection in a subject catherized with the urinary catheter system of claim 14, the method comprising: upon exposure of the pH sensor to urine for at least 24-240 hrs, detecting a colorimetric change from a first color to a second color in the pH sensor responsive to a change in urine pH from below 6-8 to 6-8 or above, and upon detection of the first colorimetric change, monitoring for a reversal of the colorimetric change from the second color back to the first color after a predetermined period of time or after treating the patient for an infection, or both.

21. The method of claim 20, wherein upon exposure of the nitrite sensor to urine for at least 24-240 hrs, detecting a colorimetric change in the nitrite sensor.

22. The method of claim 21, wherein the system comprises both the pH sensor and nitrite sensor, and if both undergo a colorimetric change, the method further comprises replacing the urinary catheter system.

23. A method for making a pH sensing membrane, the method comprising

a) dissolving polymer in a solvent to form a solution;

b) mixing an amount of at least one indicator dye in the solution;

c) mixing a lipophilic salt in the solution of step a) or mixture of step b);

d) adding a plasticizer to the mixture of step b) or c); and

e) casting an amount of the mixture of step d) onto a surface.

24. The method of claim 23, wherein the polymer comprises polyvinyl chloride, cellulose, synthetic polymer derivatives, liquid emulsions, polymer-electrolytes or ceramics.

25. The method of claim 23, wherein the polymer comprises polyvinyl chloride, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene oxide, polyhydroxyethyl

methacrylate, alginate, or cellulose acetate.

26. The method of claims 23-25, wherein the indicator dye comprises malachite green; methyl violet 10B; thymol blue; methyl yellow; bromophenol blue; congo red; methyl orange; bromocresol green; methyl red; bromocresol purple; bromothymol blue; phenol red; neutral red; naphtholphthalein; cresol red; phenolphthalein; thymolphthalein; alizarine yellow R; or Indigo Carmine; or combinations of the foregoing.

27. The method of claims 23-26, wherein the solvent comprises tetrhydrofuran; touluene; benzene; 1,1,1 Trichloroethane; xylene; carbon tetrachloride; ethyl acetate; chloroform; trichloroethylene; cellosolve® acetate; methyl ethyl ketone; acetone; diacetone alcohol; ethylene dichloride; methylene chloride; butyl cellosolve®; water; glycerol; or ethylene glycol; or a combination thereof.

28. The method of claims 23-27, wherein the lipophilic salt comprises Aliquat 336; dimethyldioctadecylammonium bromide (DDA); tridodecylmethylammonium chloride (TDMAC); or potassium tetrakis(4-chlorophenyl borate (TCPB), or a combination thereof.

29. The method of claims 23-28, wherein the plasticizer comprises O- nitrophenyloctylether (NPOE); dioctyl sebacate (DOS); bis(l-butylpentyl)adipate (BBPA); or tributyl phosphate (TBP); or a combination thereof.

30. The method of claim 23, where the at least one indicator dye comprises an amount of bromothymol blue and an amount of methyl red and/or cresol red.

31. A membrane formed by the method of any of claims 23-30.

32. An article of manufacture comprising a membrane disposed on a surface thereon, wherein the membrane comprises a composition comprising an indicator dye embedded in a hydrogel and the article comprises a catheter, a dipstick, a funnel, a dish, a container, a diaper, or an absorbent pad.

33. A method for making a pH sensing membrane, the method comprising

a) dissolving polyvinyl chloride in a solvent to form a polymer solution;

b) mixing an amount of bromothymol blue in the polymer solution;

c) mixing an amount of Aliquat 336 to the mixture of step b);

d) adding NPOE to the mixture of step c); and

e) casting an amount of the mixture of step d) onto a surface.

34. A method of detecting the presence of nitrite in a liquid sample, the method comprising obtaining a population of aggregated gold nanoparticles comprising disulfide ligands bound to the surface thereof, wherein the population of aggregated gold nanoparticles possess a first color; and

subjecting the population to an amount of the liquid sample, wherein a presence of nitrite in the liquid sample causes disaggregation of the gold nanoparticles that generates a color change from the first color to a second color.

35. The method of claim 34, wherein the aggregated gold nanoparticles are in a liquid suspension.

36. The method of claim 34, wherein the aggregated gold nanoparticles are fixated in a porous hydrogel.

37. A porous hydrogel composition comprising a population of aggregated gold

nanoparticles with disulfide ligands bound to the surface thereof.

38. An article of manufacture comprising an amount of the composition of claim 37 disposed on a surface thereof.

39. The article of manufacture of claim 38, wherein the article comprises a catheter, a dipstick, a funnel, a dish, a container, a diaper, an absorbent pad, or a catheter coupling device.

40. A method of making a composition colorimetrically responsive to nitrite, the method comprising:

c) mixing the solutions of a) and b) with an amount of plasticizer; and

d) casting an amount of the mixture of c) onto a surface.

41. The method of claim 40, wherein the polymer comprises polyvinyl chloride, cellulose, synthetic polymer derivatives, liquid emulsions, polymer-electrolytes or ceramics.

42. The method of claim 40, wherein the polymer comprises polyvinyl chloride, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene oxide, polyhydroxyethyl

methacrylate, or cellulose acetate.

43. The method of claims 40-42, wherein the solvent comprises tetrhydrofuran; touluene; benzene; 1,1,1 Trichloroethane; xylene; carbon tetrachloride; ethyl acetate; chloroform; trichloroethylene; cellosolve® acetate; methyl ethyl ketone; acetone; diacetone alcohol; ethylene dichloride; methylene chloride; butyl cellosolve®; water; glycerol; or ethylene glycol; or a combination thereof.

44. The method of claims 40-43, wherein the plasticizer comprises O- nitrophenyloctylether (NPOE); dioctyl sebacate (DOS); bis(l-butylpentyl)adipate (BBPA); or tributyl phosphate (TBP); or a combination thereof.

45. A membrane formed by the method of any of claims 40-44.

46. An article of manufacture comprising a membrane of claim 45 disposed on a surface thereon, wherein the article comprises a catheter, a dipstick, a funnel, a dish, a container, or a catheter junction device.

47. A method of making a composition colorimetrically responsive to nitrite, the method comprising:

a) Dissolving a polymer in a solvent to form a polymer solution; b) mixing an amount of gold nanoparticles in the polymer solution, and c) casting an amount of the mixture of b) onto a surface.

48. The method of claim 47, further comprising subjecting the gold nanoparticles to a functionalization step to associate a disulfide ligand to the surface of the nanoparticles, wherein the functionalization step occurs prior to step b), prior to step c) or after step c)