Methods for identifying pathological conditions in a female subject

METHODS FOR IDENTIFYING PATHOLOGICAL CONDITIONS IN A FEMALE SUBJECT

FIELD OF THE INVENTION

The present invention relates to the field of medical diagnostics and more 5 specifically, to an improved identification of secreted biological fluids using a secretion-monitoring article to identify amniotic fluid, secretions associated with bacterial, parasite, fungal, or yeast infections even in the presence of interfering biological fluids or to determine dehydration. The present invention also relates to improved methods for reducing the incidence of false positive detection.

BACKGROUND OF THE INVENTION

Many medical conditions can be diagnosed by identifying the chemical and physical properties of a body fluids, such as, by identifying salts of volatile amines in body fluids as disclosed in U.S, Pat. No. 6,099,801 and identifying the pH of urine or vaginal secretions.

Analytical procedures and devices for diagnosing medical conditions are commonly employed in assays comprising means for determining the presence and/or concentration of analytes of interest in bodily fluids with chemically reactive means adapted to provide a visual indication as a result of interacting with biological fluids, and disposable absorbent products comprising same, e.g., U.S. Pat. Nos. 4,266,022; 5,445,147 and 6,203,496. The visual indication provided by these absorbent products is not made to distinguish between indication for a substance of interest and the indication from interfering fluids, particularly, urine. Moreover, some of the articles disclosed and claimed in the aforementioned patents are directed to indicate the presence or absence of specific anlaytes in urine, e.g. for the purpose of determining dehydration as disclosed in U.S. Pat. No. 6,203,496, and thus cannot be applied also for determining medical conditions in vaginal secretions.

A number of devices involving panty shields with pH indicators are known in the art, for example in U.S. Pat. Nos, 5,217,444; 5,468,236; 5,660,790; 5,823,953;

5,910,447; 6,106,461; 6,562,297 and 6,689,114 and International patent application

WO 01/13097. However, the pH range identified by these devices is extremely narrow (e.g. US 5,660,790 and US 5,910,447) and the pH indicator composition is hydrophilic and thus cannot readily retain a stable indication, particularly indications requiring dehydrated environment. The majority of these devices can be worn by the user and whenever there is a secretion it is immediately detected by the pH indicator.

False positive readings can be caused, for example, by interfering biological fluids, such as urine. Vaginal secretions of a patient with vaginosis have a pH between

4.7 and 6.5. Because urine of a healthy patient has a pH between 5.0 and 8.0, it is very difficult to diagnose a secretion as arising from vaginosis with a high degree of confidence by just using a pH based indicator test. One solution known in the art is to sample fluid from within the vagina, where urine is not ordinarily found. This is uncomfortable and requires a visit to a health-care professional.

Another clinical situation amenable to diagnosis on the basis of pH of vaginal secretions is the identification of amniotic fluid leakage during pregnancy. If not diagnosed the amniotic sac may later rupture causing abortion of the pregnancy, or require hospitalization of the woman and infant.

U.S. Pat. No. 6,126,597 (the '597 patent) and U.S. Pat. No. 6,149,590, (the '590 patent) a continuation-in-part of the '597 patent, are directed to a device in the form of a sanitary napkin with a pH indicator configured to identify the presence of amniotic fluid in a vaginal secretion is disclosed. The '597 and '590 patents are subject to the problem of giving false positive results. The device of the '590 patent address this problem by further including in the device a microscope visualizable slide configured to gather a portion of a vaginal secretion. If the indicator shows the pH corresponding to that of amniotic fluid, the user presents a health-care professional with the slide. The health-care professional examines the slide with the help of a microscope for the typical fern-shaped patterns indicative of the presence of amniotic fluid. There are a couple of disadvantages associated with this device. First, it requires that the patient visit the health-care professional to distinguish between positive and false-positives and second, a significant amount of time is lost in the having the slide viewed by a professional to determine if amniotic fluid is actually leaking.

U.S. Pat. No. 5,897,834 discloses a device useful in a clinical setting for the 5 differentiation between urine and vaginal secretions associated with vaginosis or urine and amniotic fluid. The device includes the use of indicators with a negatively charged group immobilized to a solid polymer substrate containing quaternary ammonium groups. Further the device includes a gaseous amine-releasing reagent and an amine indicator. The use of the polymer substrate containing quaternary ammonium groups is disclosed to have an advantage of sharpening the pH dependent color transition. However, these polymer substrates have been found to be less useful in non-clinical settings: the indicated pH of dried vaginal secretions is low enough to be misdiagnosed as indicating vaginosis. Thus, although the device disclosed in U,S. Pat. No. 5,897,834 is useful in a clinical setting where the health care professional applies the vaginal secretion to the device and observes the color change, if integrated in a patient useable device, such as a panty shield, the device gives abundant false positive results.

In US patent 6,627,394, the inventors of the present invention disclosed a diagnostic device for detection of vaginosis or amniotic fluid leakage without giving a false positive result due to contact with urine, the device comprising at lease one pH indicator attached to a substrate and a reagent comprising urease attached to the substrate.

A number of devices involving swabs with pH indicators are known in the art, as disclosed for example in US Patents 6,013,036, 5,738,634, 5,664,579, 5,577,512 and 5,425,377. The advantage of using a swab substrate for pH indicators is the ability to monitor the pH of vaginal discharge directly from the vagina. As noted above, urine of a healthy patient has a pH between 5.0 and 8.0. Secretions from patients having bacterial vaginosis or parasite infection also have a pH between 4,7 and 6.5. Monitoring the. pH of vaginal discharge directly from the vagina using a swab prevents false positive results due to the contact of the pH indicator with urine. A general problem, however, with these known swab-based pH indicators is that the reaction of the pH indicator is not dependent on the buffer capacity of the secretion.

Another problem of swabs containing pH indicators is that the indicators either 5 tend to leach out from the substrate or they are not biocompatible. For example an indicator dye like Nitrazine attached to the swab tends to leach out from the substrate. Other examples of indicator materials known in the art are composed of nonbiocompatible materials. Biocompatibility is determined by means of grade zero cytotoxicity, grade zero skin irritation and grade zero sensitization according to the

USP criteria.

Still further problem with the pH indicators as are known in the art are that the color observation requires a color scale and that the sensitivity and the specificity values for detection of Bacterial Vaginosis, Candida and Trichomonas in the vagina are not optimal.

There is an unmet a need for an indicator system that can differentiate between a specific biological fluid of interest and an interfering biological fluid, such as, urine. Further there is a need for a device that can distinguish between normal vaginal secretions and those associated with amniotic fluid leakage or vaginosis. Further, a system in which false positive results are minimized and a reliable indication is stable over time, while reducing the amount of time required to get the reliable result is also needed. Such a system is ideally useable by the patient to lead to greater peace of mind and to minimize unnecessary hospital visits. The characteristics of such an indicator system must not change due to long use or as a result of a wetting drying cycle and must distinguish between interfering biological fluids and minimize false positive readings. The present invention now overcomes these problems and satisfies these needs.

SUMMARY OF THE INVENTION

The present invention overcomes the disadvantages of the prior art by the use of an indicator system integrated into various self useable products thereby providing highly specific and highly sensitive diagnostic indications, with minimal noise (interference) from nonspecific binding of interfering substances. Specificity and sensitivity of the indicator system is achieved by the unique relative amounts of the major ingredients in the system, namely, at least one indicator agent and at least one ion balance reagent.

The method of the invention can be used by the user in many forms of indicator systems, including, but not limited to, the form of a swab, gauze, panty shield, hygienic napkin, a diaper or interlabial absorbent structure. Furthermore, any user, male or female, young or old, can use the article. The particular examples of the invention as presented herein are not intended to limit the scope of the invention, but simply to illustrate and represent the numerous potential forms in which the invention can be used.

Unexpectedly, indications corresponding to pathological conditions, such as, bacterial vaginosis and amniotic fluids, are stable and remain indicative for at least 48 hours.

The inventors of the present invention discovered unexpectedly that the present indicator system reacts with secretions that have low buffer capacity (such as BV discharge) differently than with bodily fluids that have normal buffer capacity. Specifically, when the vaginal secretion possesses normal buffer capacity (commonly, vaginal secretion having ionic concentration above lOmM) such as in normal vaginal discharge, the color of the Nitrazine yellow polymer matrix changes from yellow to green/blue when the pH of the vaginal discharge is above 5. However, in cases where the buffer capacity is low (commonly, vaginal secretion having ionic concentration below lOmM) such as in bacterial vaginosis, the color of the pH indicator changes from yellow to green/blue when the pH of the vaginal discharge is in the range of 4.35.0. The color transition in pH values which are lower than pH 5 enables to reduce the percentage of false-negative bacterial vaginosis cases which may be missed when conventional immobilized pH indicators is used. Thus, the new indicator system of the present invention enables the identification of over 95% of bacterial vaginosis cases using direct testing of vaginal secretion with immediate and stable color response.

In addition to the increased sensitivity of the indicator system of the present invention in detecting vaginal discharge having low buffer capacity, the pH indicator polymer matrix of the present invention exhibits additional advantage over the art.

The pH polymer matrix of the present invention is capable of reacting differently with urine due to protonated amine cations that are present in substantial amounts only in urine, and not in the other biological fluids to be identified. Thus, although the pH of urine overlaps with the pH of amniotic fluid or BV discharge, the pH polymer matrix of the present invention is capable of differentiating between urine and amniotic fluid or BV discharge due to the presence of high amounts of protonated amine cations only in urine.

in one aspect, the present invention provides a method of identifying a pathological condition in a subject which comprises:

a, positioning a secretion-monitoring article to receive a biological fluid secreted from the subject, the secretion-monitoring article comprising a body that includes an absorbent material for absorbing the biological fluid and an indicator system having a hydrophobic chemical composition comprising an indicator agent and an ion-balance reagent which is a quaternary amine, wherein the indicator system provides an indication of physiological conditions associated with a pH or buffer capacity of the biological fluid, which indication is stable and remains indicative for at least 48 hours; and

b. viewing the article within the at least 48 hour period to identify the pathological condition based on the indication determined by the indicator system.

According to one embodiment, the pathological condition is selected from the group consisting of bacterial vaginosis, the presence of amniotic fluid and Candida.

According to another embodiment, the method further comprises any one or more of the following steps:

removing the indicator system from the article prior to the viewing step;

drying the indicator system and removing the indicator system from the article prior to the viewing step; and providing a color-encoding chart comprising a plurality of color codes and a description of medical condition for each color code; and comparing the color of said indication to the color-encoding chart, thereby determining the medical condition.

According to yet another embodiment, the ion-balance reagent and the indicator agent are present in amounts sufficient to provide a molar ratio that is within the range of 5:1 to 15:1 or within the range of 3:1 to 10:1.

According to yet another embodiment, the ion-balance reagent is in an amount 15 of about 1 % to 6% and the at least one indicator agent is in an amount of about 0.1 % to 2%, wherein the percents are weight percent based on the total weight of the composition and the total weight of the composition equals 100%.

According to yet another embodiment, the biological fluid has a pH, and the indicator agent has a pKa that is lower than the pH of the biological fluid, wherein the indicator system determines a pH which is evidence of the pathological condition.

According to yet another embodiment, the indicator system further comprises a pre-formed polymer and a plasticizer, and, optionally, a wetting agent.

According to yet another embodiment, the indicator agent is negatively charged. According to yet another embodiment, the indicator agent is a weak acid.

According to yet another embodiment, the indicator agent is selected from the group consisting of: cresol red, alizarin, bromcresol purple, chlorophenol red, nitrazine yellow, bromthymol blue bromoxylenol blue, neutral red, phenol red, thymol blue, xylenol blue and m-cresol purple.

According to yet another embodiment, the ion-balance reagent is selected from the group consisting of: di(alkyl)dimethyl ammonium chloride, N-methyI-N,Nbis(alkanoyl oxyethyl)-N-(2-hydroxyethyl) ammonium methy I sulfate, vinylbenzyl dimethylcocoammonium chloride, methyl trioctyl ammonium chloride, tricaprylylmethyl ammonium chloride, tridodecylmethyl ammonium chloride, cetyltrimethyl ammonium chloride and combinations thereof.

According to yet another embodiment, the method of the invention provides a stable indication, specifically, the method of the invention provides an indication that is stable for at feast 72 hours so that the article can be viewed within the at least 72 hour period to identify the pathological condition based on the pH that is determined by the indicator system.

According to yet another embodiment, the indicator system irreversibly changes color for at least 48 hours when contacted by a vaginal secretion associated with vaginosis or amniotic fluid leakage. According to yet another embodiment, the indicator system reversibly changes color when contacted by urine.

According to yet another embodiment, the absorbent material is selected from the group consisting of a swab, gauze, panty shield, hygienic napkin, diaper and interlabial absorbent structure.

According to yet another embodiment the indicator system further comprises a 20 pre-formed polymer in an amount of about 20% to 50%; a plasticizer in an amount of about 15% to 40%; and a wetting agent in an amount of about 15% to 45%; wherein the ion-balance reagent is present in an amount of about 0.1% to 10%; and the indicator agent is present in an amount of about 0.05% to 5%; wherein the percents are weight percent based on the total weight of the indicator system and the total weight of the indicator system equals 100% and the indicator system provides an indication of physiological conditions associated with the pH or the buffer capacities of the biological fluid.

According to yet another embodiment, the pre-formed polymer is present in an amount of about 36% to 39%; the plasticizer is present in an amount of about 27% to 29%; the wetting agent is present in an amount of about 29% to 31%; the ionbalance reagent is present in an amount of about 4% to 6%; and the indicator agent is present in an amount of about 0.2% to 0.4%.

According to yet another embodiment, the pre-formed polymer is a weak 5 acidic polyelectrolyte polymer, cellulose, sodium carboxymethyl cellulose, ethyl cellulose, nitrocellulose, cellulose acetate and combinations thereof.

According to yet another embodiment, the wetting agent is selected from the group consisting of: triethylene glycol, ethylene glycol, sorbitol, 2-ethoxy ethanol and combinations thereof.

According to yet another embodiment, the plasticizer is selected from the group consisting of: castor oil, diacetylated monoglycerides, diethyl phthalate, glycerin, mono- and di-acetylated monoglycerides, polyethylene glycol, propylene glycol, triacetin, triethyl citrate, bis-(2-butoxyethyl) adipate, bis-(2-ethylhexyl) sebacate, dibutylphthalate and combinations thereof.

According to yet another embodiment, the pre-formed polymer is cellulose acetate in an amount of about 20% to 30%; the plasticizer is dibutylphtalate in an amount of about 10% to 20%; the wetting agent is an ethoxy ethanol polymer in an amount of about 20% to 30%; the ion reagent is a quaternary amine in an amount of about 1% to 10%; and the indicator agent is nitrazine yellow in an amount of about

0.05% to 5%; wherein the percents are weight percent based on the total weight of the mixture and the total weigh of the mixture equals 100%.

According to yet another embodiment, the indicator system further comprises a solvent in an amount of about 1 to 30 ml of solvent for each 150 mg of pH indicator mixture.

These and further embodiments will be apparent from the detailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the color change of the polymer versus the change in pH, wherein the color scale conversion is as follows: 0- Yellow; 1- Light

Green; 2- Green; 3- Dark green.

Figure 2 is a graph demonstrating the buffering capacity of vaginal secretions.

The graph compares the pH of the butter or secretion being titrated by the NaOH 0.1 M added.

Figure 3 is a general schematic perspective view of a different embodiment designed to be integrated on ordinary panty shield.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method for using a secretion-monitoring article that allows both professional and untrained users to monitor secreted biological fluids, such as secretions associated with bacterial vaginosis, with high accuracy.

Bacterial vaginosis (BV) is characterized by production of increased quantities of malodorous vaginal discharge. As mentioned above, one of the characteristics of BV is the homogeneous discharge. A women having BV typically has an increase in the discharge amount. The source of this liquid is extracellular fluid (interstitial fluid) that surrounds the epithelial cells in the vagina wall. A decrease in protein levels and other large organic molecules and the increase of water content in BV secretions lowers the buffer capacity of the secretions. Thus, secretions associated with BV have a lower buffer capacity than normal vaginal secretions.

The pH indicator polymer matrix of the present invention possesses significant advantages over the art. The inventors of the present invention discovered unexpectedly that the present indicator system reacts with secretions that have low buffer capacity (such as BV discharge) differently than with bodily fluids that have normal buffer capacity. Thus, in cases where the buffer capacity is low (commonly, vaginal secretion having ionic concentration below lOmM) such as in bacterial vaginosis, the color of the pH indicator changes when the pH of the vaginal discharge is in the range of 4.3-5.0.

According to one embodiment, the pathological condition is selected from the group consisting of bacterial vaginosis, the presence of amniotic fluid and Candida. It is standard practice, for patients with vaginal symptoms, to rule-out BV and Trichomonisis and treat for Candida. As the color change caused by vaginal secretions infected with Candia is similar to the color change obtained with normal vaginal secretion, the article of the invention may be used to distinguish vaginal secretions infected with Candia from vaginal secretions infected with BV and Trichomonisis.

The article of the invention thus provides a simple test for eliminating BV and Trichomonisis from consideration thereby providing treatment merely for Candida.

Furthermore, the pH indicator polymer matrix consists of a special composition that reacts with fluids containing protonated amine cations, such as urine, in a different way than it reacts to other biological fluids that have a low concentration of protonated amine cations, such as amniotic fluid. Specifically, during the drying process of the indicator system, the turquoise or green color obtained following the contact of urine with the indicator fades as it dries and the color of the indicator becomes yellow¹ again. In contrast, when the indicator is contacted by amniotic fluid the indicator strip changes color from yellow to green or turquoise and does not fade when dried.

Furthermore, the polymer pH indicator of the present invention reacts with normal urine differently than infected or protein-containing urine, In one non-limiting example, the indicator reacts with normal urine (pH 5-8), which changes the color from yellow to green or turquoise, During the drying process the color change of the indicator that has been contacted with normal urine fades as it dries and becomes yellow again. In contrast, when the indicator is contacted by infected or protein containing urine the indicator strip changes color from yellow to green or turquoise and does not fade when dried. Advantageously, this embodiment is well suited for all types of use, for example in pediatrics, geriatrics, and gynecology, and could be presented to the user in many forms.

The secretion-monitoring article can be implemented using many devices and methods. The body of the secretion-monitoring article of the present invention comprising the absorbent material can be supplied to the user for example in the form of a pad, gauze, a swab, a fiber ball, a sanitary napkin, diaper, panty shield, or interlabial structure. Details of manufacture of these are well known to one skilled and have been fully described in the prior art, for example U.S. Pat. Nos. 5,217,444, 5,897,834, and 6,149,590.

The indicator system utilized by the method of the present invention is capable of providing a visible indication which is stable for at least 48 hours, or even for at least 72 hours.

The terms stable indication and irreversible indication are interchangeably used herein to describe an indication, typically a color indication, that once obtained remains sufficiently unaltered for a time sufficient for clinical examination by a professional. The color change may be stable for at least 48 hours, or at least 72 hours, and in some embodiments, the color change is stable for about a week.

Accurate, absolute and fast determination of a medical condition is extremely important in various medical conditions, including, vaginal infections and amniotic leakage. As detailed above, a number of compositions and devices comprising same, having indicators for indicating medical conditions are known in the art. However, they often provide false positives due to changes in pH on drying, the presence of interfering biological fluids or due to repetitive drying/wetting cycles. Vaginal infections and amniotic leakage are particular medical conditions that can be diagnosed using the articles known in the art, however, there are often misdiagnosed due to the plurality of substances having similar pH levels, which are commonly present in vaginal secretions.

Inaccurate diagnosis of vaginal infections and amniotic leakage due to false positive readings is stressful and time consuming to the user. Amniotic fluid leaking from the vagina of a pregnant woman may occur during pregnancy when the amniotic sac integrity is compromised and a small amount of amniotic fluid may leak out through the cervix and from the vagina. If diagnosed as such, measures such as patient rest or sealing of the amniotic sack using biological glue may be prescribed. If not diagnosed, the amniotic sack may later rupture causing abortion of the pregnancy, or require hospitalization of the woman and infant. If the infant is born prematurely, death or severe handicap may be a result. Extended hospitalization of the infant in an incubator is often necessary,

Due to the severe consequences of amniotic fluid leakage, pregnant women undergo severe stress and often go to a health-care professional upon secretion of any liquid from the vicinity of the vagina. The health-care professional looks for the presence of amniotic fluid by checking the pH of the vaginal secretions, amniotic fluid having a pl-1 of between 6.0 and 8.0. Since pregnant women often have urinary incontinence and since urine typically has a pH of between 5.0 and 8.0, if only pH is checked, a false positive result may occur: urine being identified as amniotic fluid, Consequently, it is necessary that such a vaginal secretion be examined using a microscope for the presence of a fern-shaped pattern indicative of amniotic fluid.

As the time between the fluid secretion and the arrival at the health-care professional may be long, there is often no evidence of amniotic fluid upon examination. The secretion may mistakenly be assumed to be urine, often with tragic consequences. On the other hand, the healthcare professional may decide to err on the side of caution, misdiagnosing the secretion of urine as amniotic fluid leading to an unnecessary hospitalization and patient stress.

Commonly, false positive readings of vaginal secretions are caused due to the presence of urine. The pH of vaginal secretions of a patient having bacterial vaginosis is between 4.7 and 6.5. The pH of urine of a healthy patient is within the range of 5.0 and 8.0. Thus, diagnosing bacterial vaginosis with a high degree of confidence cannot be relied upon pH-based test, unless the sample fluid is collected directly from the vagina, where urine is not ordinarily found. However, such examination is uncomfortable and requires intervention of a health-care professional.

The method of the invention provides an accurate determination of a medical condition due to the unique content of the indicator system, namely, a pre-formed polymer, at least one indicator reagent, and at least one ion-balance reagent.

In one embodiment of the invention, the secretion-monitoring article comprises a body that includes an absorbent material for absorbing a biological fluid secreted from a person and an indicator system comprising at least one indicator agent that reacts with biological fluid differently. One example of such an indicator agent is a composition that reacts with fluids containing protonated amine cations, such as urine, in a different way than it reacts to other biological fluids that have a low concentration of protonated amine cations, such as amniotic fluid.

The article used for the method of the invention can be embodied as a swab, gauze, shield, hygienic napkin, diaper or interlabial absorbent structure and can be used to indicate, the presence of abnormal ammonium concentration in human urine, amniotic fluid leakage, or biogenic secretions associated with bacterial vaginosis, parasite infections, or deficiency of lactobacillus population, without giving a false positive result.

In another embodiment of the invention, the secretion-monitoring article comprises a body that includes an absorbent material for absorbing a biological fluid secreted from a person and an indicator system comprising an indicator mixture that reacts with normal urine differently than infected or protein containing urine. In one non-limiting example, the indicator reacts with normal urine (pH 5-8), which changes the color from yellow to green or turquoise. During the drying process the color change of the indicator that has been contacted with normal urine fades as it dries and becomes yellow again. In contrast, when the indicator is contacted by infected or protein containing urine the indicator strip changes color from yellow to green or turquoise and does not fade when dried. Advantageously, this embodiment is well suited for all types of use, for example in pediatrics, geriatrics, and gynecology, and could be presented to the user in many forms, such as, as a diaper or a panty liner.

The pH determining member of the indication system can be any pH determining device, for example as a color changing indicator (e.g., litmus paper) or a mobile pH probe. In one embodiment, the pH determining member is a color changing indicator, such as a pH determining member made from the pH indicator mixture described herein below. More than one pH determining member can be part of the indicator system. The pH determining members should be capable of determining substantially different pH ranges or capable of reacting differently to different biological fluids to produce a different color change.

According to one embodiment of the present invention, the indicating composition is made with nitrazine yellow that indicates the presence of a fluid with a pH of around 4.2 to 7.0. Upon contacting vaginal secretions having a pH of 5.2 or greater, the color changes from pale yellow to blue or green, which indicates possible BV or Trichomonas. At pH of 5.1 or lower, but greater than 4.2, the color change depends on the ionic strength of the vaginal discharge: the more fluidic is the discharge; the change in color is less evident. Fluids with pH levels of 4,2 or lower do not cause a change in the color of the indicating composition.

According to another embodiment the secretion-monitoring article includes mounting means for positioning the absorbent body to receive the fluids secreted from a subject (the user) in need thereof during the normal activity of the user, such mounting means being, for example, an adhesive strip or other attachment member.

An example of a mounting means that is well known in the art is an adhesive strips associated with the article, The article may further comprise one or more adhesive strips. The user removes the release tape to expose the adhesive strip of the article and places the article in the crotch portion of their undergarment. This prevents the article from moving out of position during regular use. Types of adhesive compounds thafcan be used are well known in the art.

The pH indicator polymer matrix is impregnated onto a substrate which can be made of many materials, for example, polypropylene, paper or cotton, polyester membranes and can be of many structures including of a membrane, fabric, mesh, gauze, thread, fiber and a sheet. The polymer matrix pH indicator contains a mixture of pre-formed polymer (such as cellulose), a plasticizer, a wetting agent, an ionbalance reagent and an indicator, In some cases a solvent is added to the indicator mixture, it is also possible that the polymer solution is polymerized after the addition of the plasticizer, the wetting agent, the ion-balance reagent and the indicator to the mixture, The mixture may be applied onto a substrate by dipping the substrate in the mixture or by spraying or spreading the mixture onto the substrate. The substrate with the applied mixture is allowed to dry. When dry, the polymeric pH indicator is bound to the substrate.

In one embodiment according to the present invention, a pH indicator is mixed with a polymer in a suitable solution and then applied to a substrate. According to specific embodiments, the polymer solution is prepared containing pre-formed polymer, plasticizer, a wetting agent, an ion-balance reagent, a solvent and an indicator.

Any non-toxic suitable plasticizer can be used, such as, bis-(2-ethylhexyl) sebacate (DOS, CAS 122-62-3), diethyl phthalate (DEP, CAS 84-66-2), bis-(2butoxyethyl) adipate (BBPA, CAS 141-18-4), dibutyl phthalate (DBP, CAS 84-74-2) and dioctyl phthalate (DOP, CAS 117-81-7). The plasticizer makes up 15% to 40% of the weight ofthe solution, 20% to 35% ofthe solution, 21% to 31% of the solution, and 27% to 29% by weight of the solution, As is clear to one skilled in the art, it is also possible to use a combination of suitable plasticizers when making the polymer solution.

Any suitable volatile wetting agent can be used, such as, triethylene glycol (CAS 112-27-6), ethylene glycol (CAS 107-21-1), or 2-ethoxy ethanol (CAS 110-805). The wetting agent makes up 15% to 45% of the weight of the solution, 21% to 40% of the solution, 26% to 39% of the solution, or 29% to 31% by weight of the solution. As is clear to one skilled in the art, it is also possible to use a combination of suitable wetting agents when making the polymer solution.

Any suitable ion-balance reagent can be used, such as, tridodecylmethyl 5 ammonium chloride (TDMAC1, CAS 7173-54-8), tricaprylylmethyl ammonium chloride (Aliquat 336, CAS 5137-55-3), or cetyltimethyl ammonium chloride (CTAC, CAS 112-02-7). The ion-balance reagent makes up 0.1% to 10% of the weight of the solution, 0.5 % to 8% of the solution, 0.8% to 7% of the solution, or 4% to 6% by weight of the solution. As is clear to one skilled in the art, it is also possible to use a combination of suitable ion-balance reagents when making the polymer solution.

The polymer used in the pH indicator polymer matrix solution is commonly suitable for a pad, gauze, a fiber ball, a sanitary napkin, diaper, panty shield, and interlabial structure and may be selected from various preformed polymers, such as, nitrocellulose (CAS 9004-70-0), cellulose acetate (CAS 9004-35-7) or ethyl cellulose (CAS 9004-57-3). The preformed polymer makes up 20% to 50% of the weight of the solution, 25% to 45% of the solution, 30% to 43% of the solution, or 36% to 39% by weight of the solution. As is clear to one skilled in the art, it is also possible to use a combination of suitable preformed polymers when making one polymer solution.

It is to be understood that the actual amounts of the components of the solution are added so that the sum of weights of pre-formed polymer, plasticizer, wetting agent and ion-balance reagent is equal to 100%.

Following the preparation of the polymer solution, the desired indicator is added to the solution. Any suitable indicator can be used, including, but not limited to, a negatively charged functional group such as acetate or sulfonate. The indicators used separately or in combination, are chosen from amongst indicators listed in Table 1 and in US Patent 5,897,834. The total amount of indicator added is 0.05% to 5% of the weight of the polymer solution as described above, 0.05% to 3% of the polymer solution, 0.1% to 1% of the polymer solution, and 0.2% to 0.5% of weight of the polymer solution.

The term weight percent as used herein refer to the weight percent of the components of the indicating composition before adding the solvent and before drying,

i.e. before reaching the final dry composition. The final dry composition typically does not contain any of the volatile components, namely, water, solvent and the volatile wetting reagent (e.g. 2-Ethoxy ethanol). The volatile components evaporate during the manufacturing process.

Any suitable solvent or mixture of solvents may be used for preparing the pH 10 indicator polymer matrix solution, including, but not limited to acetone, ethyl acetate or substantially volatile ethers such as diethyl ether, isopropyl ether, t-butyl methyl methyl ether or Tetrahydro furan. The amount of solvent added to the solution is suitable for making any easily applied solution/indicator polymer matrix. Typically, 900 mg of preformed polymer is dissolved in between 30 ml and 110 ml of solvent, or between 50 ml and 100 ml solvent.

Once the pH indicator polymer matrix is ready, it is applied by suitable means to the substrate. The mixture may be applied on the substrate by spraying or spreading, or by dipping the substrate in the pH indicator polymer matrix. The substrate can be of many suitable materials known in the art such as polyester membranes or fabrics, polypropylene membranes, cellulose membranes, paper, cotton or linen. The structure of the substrate may be for example, a fiber, a mesh, gauze, a fabric or a membrane. The solvent of the mixture is allowed to evaporate.

Although some of the compounds of the polymer solution are known as toxic according to their MSDS (material safety data sheet), once the mixture impregnate onto the substrate, for implementing the secretion monitoring article the final product is non toxic, non leaching and biocompatible with grade zero cytotoxicity, grade zero sensitization and grade zero irritation.

It is imperative that there be substantially no leaching of the pH indicator polymer matrix components from the substrate to which the indicator system is impregnated. The attachment of indicators to a substrate is well within the ability of one skilled in the art. Chemical compounds that are suitable for use as an indicator to be part of the polymer matrix indication system are solfonephthaleine series or Azo dyes. Suitable indicators include Nitrazine yellow, thymol blue, bromthymol blue, xylenol blue, bromoxylenol blue, phenol red, m-cresol purple, chlorophenol red, bromcresol purple, alizarin, neutral red, and cresol red, see Table 1. A list of other suitable indicators can be found, for example, in US Patent 5,897,834. It is clear to one skilled in the art that the indicators specifically mentioned herein are just examples and any suitable indicators may be used.

TABLE 1:

<td> Indicator</td><td> Aqueous pH transition range</td><td> Color change</td><td> CAS</td>

<td> 1. Cresol lied</td><td> 7.2 - 8.8</td><td> Yellow to reddish purple</td><td> 1733-12-6</td>

<td> 2. Alizarin</td><td> 5.5 - 6.8</td><td> Yellow to violet</td><td> 72-48-0</td>

<td> 3. Bromcresol Purple</td><td> 5.2-6.8</td><td> Yellow to purple</td><td> 115-40-2</td>

<td> 4. Chlorophenol Red</td><td> 5,2-8.8</td><td> Yellow to red</td><td> 4430-20-0</td>

<td> 5. Nitrazine Yellow</td><td> 6.0-7.2</td><td> Yellow to bright blue</td><td> 5423-07-4</td>

<td> 6. Bromthymol Blue</td><td> 6.0-7.6</td><td> Yellow lo blue</td><td> 34722-90-2</td>

<td> 7. Bromoxylenol Blue</td><td> 6.0-7.6</td><td> Yellow to blue</td><td> 40070-59-5</td>

<td> 8. Neutral Red</td><td> 6.8-8.0</td><td> Red to yellow</td><td> 553-24-9</td>

<td> 9. Phenol Red</td><td> 6.8-8.2</td><td> Yellow to red</td><td> 34487-61-1</td>

<td> 10. Thymol Blue</td><td> 8.0 - 9.2</td><td> Yellow to blue</td><td> 81012-93-3</td>

<td> 11. Xylenol Blue</td><td> 8.0-9.6</td><td> Yellow to blue</td><td> 125-31-5</td>

<td> 12. m-Cresol purple</td><td> 7.4-9.0</td><td> Yellow to purple</td><td> 2303-01-7</td>

According to the method of the invention, an article is attached to the underwear ofthe user such that the indicator strip faces the vagina. Prior to contacting vaginal fluids the color of the indicating composition is pale yellow. Following accumulation of vaginal secretion on the indicator strip, the user removes the article from her underwear and removes the indicator strip from the article. The indicator strip is left to dry for several minutes. For this purpose, the indicator strip may be left to dry at room temperature for about 2 to about 10 minutes. The user than views the color of the indicating strip and interprets the results in accordance with the following color-coding description;

Blue-green color - indicates possible BV (Bacterial Vaginosis) or Trichomonas. Upon appearance of blue color the patient should consult a physician. If the change in color is caused by urine and not by BV than the blue-green color fades away within less than 10 minutes.

Yellow color - indicates low probability of BV or Trichomonas. In such cases, the cause of the vaginal secretion may be yeast or transient irritation.

Upon contacting vaginal secretions having a pH of 5.2 or greater, the color changes from pale yellow to blue or green. At pH of 5.1 or lower, but greater than 4.2, the color change depends on the ionic strength of the vaginal discharge: the more fluidic is the discharge, the change in color is less evident. Fluids with pH levels of

4.2 or lower do not cause a change in the color of the indicating strip.

In the event that the indicator strip stains blue or green, the discharge acidity parameters are disordered and the risk of bacterial or parasitic infection is high; if the indicator strip does not change color or is initially stained blue or green but the color fades back to yellow during the 10 minute drying period, then there is a low probability of bacterial or parasitic infections and a high probability that the discharge was in fact urine.

For identifying leakage of amniotic fluids according to the method of the present invention, an article is attached to the underwear of the pregnant woman as described above. Prior to contacting any fluids the color of the indicating composition is pale yellow. Following wetness sensation and accumulation of fluid on the indicator strip, the pregnant woman removes the article from her underwear and removes the indicator strip from the article. The indicator strip is left to dry for several minutes. For this purpose, the indicator strip may be left to dry at room temperature for about 2 to about 30 minutes. The pregnant woman than views the color of the indicating strip and interprets the results in accordance with the following color-coding description:

Blue-green color - indicates that the pregnant woman may be leaking amniotic fluid. Upon appearance of the blue-green color the patient should contact her healthcare provider without delay. If the change in color is caused by urine and not by amniotic fluid, than the blue-green stain fades away within less than 30 minutes.

Yellow color - indicates that the wetness was probably not caused by amniotic fluid. In such cases, the cause of the wetness sensation was probably urine. Amniotic fluid normally has pH levels varying between 6.5 and 7.5 units.

Urine has pi-1 levels between 5.0 and 8.0 units. Upon contacting fluids having a pH of 5 5.2 or greater, the color changes from pale yellow to blue-green. Stains observed on the strip can be caused by either amniotic fluid leak or by urine. After drying up to 30 minutes, stains caused by urine fade back to yellow, while stains caused by amniotic fluid remain stable.

The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

EXAMPLE 1: Preparation of the secretion-testing swab containing the Nitrazine yellow pH indicator polymer matrix.

Method of Preparation

1) Polymer matrix indicator:

Step 1: Dissolving cellulose acetate 24% to 28% in Acetone.

Step 2: Adding to the solution Dioctyl Phthalate 4%-22%.

Step 3: Adding to the solution Tridodecylmethyl ammonium chloride l%to5%.

Step 4: Adding to the solution 2-ethoxyethanol 24% to 28%.

Step 5: Adding to the solution Nitrazine Yellow 0.2% to 0.5% dissolved in double distilled water 24%-28%.

Step 6: Impregnating the solution to a swab or strip.

Example 2; Color change of the Nitrazine yellow pH indicator polymer matrix in different buffer capacities.

lOOmM buffer phosphate citrate was prepared with seven different pH values.

Each buffer was diluted to four different concentrations: 50, 20, 10 and 5 mM and the pH were adjusted using NaOH 1M or HCI 1M. A Nitrazine yellow polymer matrix of the invention was dipped in each buffer and the change in color was noted and represented by a numeric values as follows: 0 = Yellow; 1= Light Green; 2= Green; 3= Dark green. As revealed from the results summarized in Table 2 and Figure 1, when the solution has ionic concentration of 50mM or lOOmM, the Nitrazine yellow pH indicator polymer matrix changed the color from yellow to green/blue when the pH is above 5. In cases where the ionic concentration is lower than 20mM, the color change occurs in a dynamic pH range of 4.3-5.0.

TABLE 2

<td> pH (±0.05)</td><td> 100 mM</td><td> 50 mM</td><td> 20 mM</td><td> 10 mM</td><td> 5 mM</td>

<td> 4.0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 4.3</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 1</td>

<td> 4.5</td><td> 0</td><td> 0</td><td> 0</td><td> 1</td><td> 1</td>

<td> 4.7</td><td> 0</td><td> 0</td><td> 1</td><td> 1</td><td> 2</td>

<td> 5.0</td><td> I</td><td> 1</td><td> 1</td><td> 2</td><td> 2</td>

<td> 5.2</td><td> 1</td><td> 1</td><td> 2</td><td> 2</td><td> 2</td>

<td> 5.5</td><td> 2</td><td> 2</td><td> 3</td><td> 3</td><td> 3</td>

<td colspan="5"> *Color scale conversion: 0- Yellow; 1- Light Green; 2- Green; 3- Dar</td><td> &lt; green;</td>

A follow-up experiment was done using commercially available Nitrazine 20 Paper (APOTHECON Inc., Princeton, New Jersey) with the same regime of pH buffer solutions. The Nitrazine Paper did not significantly change color in any of the pH buffer solutions. The results indicated that the commercially available Nitrazine Paper is not sensitive, enough to distinguish differences in pH of any of the measured solutions.

An explanation for the fact that the Nitrazine yellow pH indicator polymer matrix of the present invention changed the color from yellow to green/blue when the pH is above 5 only in solutions with high buffer capacity while in cases where the solution possesses low buffer capacity, the color change occurs in a pH ranging between 4.3-5.0 is detailed hereinbelow.

The commercial Nitrazine yellow is a weak acid pH indicator, which when 10 dissolved in water dissociate slightly and form the conjugate base described below. The chemical composition of the commercial Nitrazine paper is hydrophilic, so that it contains some degree of ionic buffer capacity. Thus, the color change in the commercial Nitrazine yellow is independent on the buffer capacity of the solution and appears in pH 5 even in solutions with low buffer capacity.

Commercial Nitrazine yellow indicator:

HIn (aq) + H₂O H₃O⁺ (aq) + In- Acid Conjugate base (Yellow) (Blue)

[Equation 1]

In contrast to the commercial indicator, the Nitrazine yellow pH indicator polymer matrix of the present invention is hydrophobic since it is composed of an organic substrate, thus it does not have ionic buffer capacity. Therefore, using the pH indicator polymer matrix of the present invention, it is possible to obtain color change in pH lower than 5 in solutions with low buffer capacity. Thus, the pH indicator is dependent on the buffer capacity of the solution.

The mechanism of color change of the Nitrazine yellow pH indicator polymer matrix is described in the following equation (Nitrazine yellow-NY):

ROH + NV + OH‘ t; RONR) + H₂O

NY-Yellow ion balance reagent Base NY complex - Blue Water

[Equation 2]

The reaction is in equilibrium and the color of the polymer depends on the ratio of free NY compared to the NY complex.

It is clear from the results (Table 2) that when the buffer capacity of the 5 solution is lower the color change occurs at a lower pH. The variation between the first color change in the 5 mM buffer solution is approximately 0.7 pH units lower than the first color change of the 50 mM buffer solution.

This is achieved in the polymer matrix of the invention indicator because of its unique formulation. The nitrazine yellow (NY) environment in the polymer matrix is hydrophobic, composed of electronic neutral organic substrate except of the ion balance reagent that does not contribute to the acid-base balance. In contrast, the chemical composition of the commercial nitrazine Paper is a hydrophilic formulation that contains some degree of ionic buffer.

Example 3: Assessment of the buffer capacity of vaginal secretions:

In order to examine the buffer capacity of vaginal secretions, different buffer solutions were titrated with 0.1 N NaOH in comparison with vaginal secretions.

The vaginal secretions were collected with a sterile swab. The sterile swab was weighted on an analytical balance before and after secretion sampling. The secretion was then diluted in ddPHO and titrated as the other buffer solutions with NaOH 0.1M. The dilution factor was taken in account in determining the buffer capacity of the vaginal secretion. The results of the titrations are summarized in Figure 2.

The results presented in Figures 1 and 2 demonstrate that the Nitrazine yellow pH indicator polymer matrix of the present invention has an advantage compared to the commercial Nitrazine paper in detecting vaginal infections having low buffer capacity characteristics. Specifically, infected vaginal secretions having pH between

4.3-4.9 and low buffer capacity will be detected only by the pH indicator polymer matrix of the present invention.

Example 4: A panty shield capable to distinguish accurately between an amniotic fluid leak and wetness caused by urine incontinence

The article can be a sticker or a pantyliner with an embedded indicator strip.

The strip contains the pH indicator Nitrazine-yellow which has a pKa of 6.6 in aqueous solution.

Figure 3 is a general schematic perspective view of a panty shield (32) with a micro-porous top layer (34) with conically shaped holes (42) that by dripping by pipette (38) a drop (40) of any liquid, the drop will be absorbed(44) in one side ofthe micro-pours layer (34) and will not get out. Like any ordinary panty shield (32) it has an absorbent layer (36) and might have wings (46) to support the attachment to the woman’s panty.

Reaction ofthe indicator with amniotic fluid (pH 6-8) changes the color from yellow to stable dark blue. Reaction of the indicator with urine (pH 5-8) changes the color to fading green or fading turquoise. Urine with lower pH 5-5.5 doesn’t change the indicator color.

The difference between the color reaction of the indicator with amniotic fluid and with urine consists of two parameters: the chemical composition of the fluids and the indicator’s polymer chemical structure.

The following two equations demonstrates the different reactions

[Equation 3]: R—OH + X' + NR?CF 5 R—0 * NR?+H₂O + CF Yellow Ion balance Ion pair

Reagent Blue

[Equation 4]: R—OH + X’ + NH?CF S R— 0 ’ NH? +H₂O + CF Yellow Ammonium Ion pair

Turquoise

KEY:

X' = Base; NR? = Ion balance reagent; R— 0 ’ NR? =- phenolate - ion-balance reagent 25

The ratio of ion-balance reagent versus indicator in the polymer matrix controls the transition point of the color and the color stability while drying. In the drying process the ion pair phenolate (the active site of the indicator) - ion-balance reagent is stable, which cause a stable performance of the color (equation 3 - the relative concentration of the component does not change). In a different case where the concentration of the ion-balance reagent in the polymer is higher, the color of the indicator is getting dark while drying. The color darkening while drying is due to continuance deprotonation of the indicator’s phenol by the basic excess of the ion balance reagent (equation 3- while drying the base concentration is getting high and the equilibrium turned to the right). The optimum molar ratio of ion-balance reagent to indicator is within the range of 5:1 to 15:1; 3:1 to 10:1 or about 10:1.

Ammonium ions in solution react like the ion-balance reagent and compete with the phenolate active site. While drying the ion pair phenolate - Ammonium hydrolyzed spontaneously to give the protonated yellow phenol (equation 4) while the phenolate - ion-balance reagent pair is stable (equation 3).

In a case where the medium contain ammonium ion the color changes govern by the relative concentration of the ion balance reagent in the polymer and the ammonium ion in the medium.

For example: in lOOmM buffer solution that contains 25mM ammonium ion the concentration of the ammonium is in two orders of magnitude higher than the ion balance reagent in the polymer. These differences govern the turquoise color in solution and the fading color on drying.

Urine contains ammonium ions in concentration of 30 - 50 mM; amniotic fluid does not contain any substantial amount of ammonium ions, thus causing no fading influence as urine does.

Method of Preparation:

Step 1: To a 5 ml of Acetone add 150 mg Cellulose acetate, 107 μΐ Dibutylphthalate, 23μ1 Aliquat, 150 μΐ 2-Ethoxy ethanol and 2.4 mg Nitrazine yellow dissolved in 150 μΐ DDW.

Step 2: Stir the mixture for few minutes to complete dissolving.

Step 3: Coat a polyester monofilament screening fabric with the polymer solution to give the desired product.

Example 5: A device able to distinguish accurately between normal urine 10 and infected urine

It is to be understood that the term normal urine as used herein refers to urine having normal ammonium concentration and a pH ranging from about 5 to about 8. By contrast, urine of a dehydrated subject has abnormal ammonium concentration, typically, above 60 mM and an increased specific gravity.

The reoccurrence of urinary tract infections in certain patients present the need to quickly and easily diagnose whether the patient has another urinary tract infection. Presently, to determine if a patient has a urinary tract infection they must make an appointment to¹ visit a doctor. Furthermore, if the patient is susceptible to the reoccurrence of urinary tract infections they must make periodic visits to the doctor’s office to ensure that the infection has not reoccurred. Having a device that would allow the user to determine if they had a urinary tract infection again would minimize stress and time consumed by visits to the doctor’s office and result in quicker diagnosis of the.infection, resulting in a reduction in pain suffered by the patient and a more timely treatment of the infection.

The article in this example is a diaper or a panty liner with an indicator that can distinguish between normal urine and infected urine. The user wears the article so that urine can come in contact with the article. The reaction of the indicator with urine (pH 5-8) changes the color from yellow to green or turquoise. The drying process ofthe indicator strip at room temperature is short (10 minutes). When normal urine conies in contact with the indicator strip the color changes fade while drying. The color change is completely reversible and the strip becomes yellow again. On the other hand when infected urine comes in contact with the indicator strip the color changes to green or turquoise and stay constant while drying.

The reversibility of the color changes depends on two different factors of the environment:

1. Chemical environment:

a. The pH level of the fluid - pH level higher than the pKa gives a stable color change.

b. Buffer capacity of the solution c. Ammonium salts content in the solution .

2. Biological environment:

a. Protein presence in urine gives a stable color change and the reaction is not reversible.

Infected urine provides a stable color change to the indicator, which color change is not reversible, Furthermore, bacteria presence in vaginal secretion fluid also gives a stable color change so that the color change is not reversible.

EXAMPLE 6 - Stability of indication I

Solution A: 370 mg cellulose acetate, 280 mg DBP, 150 mg sorbitol, 150 mg

2-ethoxyethanol', 50 mg TDMAC were combined. 3 mg Bromthymol blue were added, 20 ml THF were added. The solution was vigorously stirred.

Solution B: 370 mg cellulose acetate, 280 mg BBPA, 300 mg ethylene glycol, mg TDMAC were combined. 3 mg m-cresol purple and 30 units urease were added. 20 ml 20 THF were added. The solution was vigorously stirred.

Solution C: lOmM buffer Citrate Phosphate solution at different pH values ranging from 3.0 to 7.0. The lOmM buffer solution was obtained by diluting lOOmM buffer solution. The different pH levels are result of mixtures at different ratios between the buffer components (lOOmM Citric acid and 200mM dibasic Sodium

Phosphate) adjusted to the final pH values with 1M Hydrochloric Acid or 1M Sodium Hydroxide solutions.

Solution D: 0.1% BSA in double distilled water and pH 4.5 adjusted to the 5 final pH values with 1M Hydrochloric Acid or 1M Sodium Hydroxide solutions.

(la) Cotton gauze was dipped in Solution A. When the solution dried, the cotton gauze was cut in half. The first half was dipped in a pH 7 test solution. The first half became purple. The first half was allowed to dry in ambient conditions, with no substantial change of color. After three hours, the second half was dipped in a pH 7 test solution. The second half became purple. The colors of the first half and of the second half were substantially the same.

(lb) Cotton gauze was dipped in Solution B. When the solution dried, the cotton gauze was cut in half. The first half was dipped in urine. The first half became violet. The first half was allowed to dry in ambient conditions, with no substantial change of color. After three hours, the second half was dipped in urine. The second half became violet. The colors of the first half and of the second half were substantially the same.

(lc) Solution A and Solution B were applied in alternating stripes on cotton gauze at a density of about 50 μΐ/mm . Amniotic fluid was applied to the gauze, changing the color of the stripes of Solution A to purple. Urine was applied to the gauze, changing the color of the stripes of Solution B to violet. The gauze was allowed to dry at ambient conditions for three hours and cut in half. Urine was applied to the first half. The colors of the stripes in the first half and the second half ofthe gauze were substantially the same.

(ld) Strips coated with solutions A or B were dipped in Solutions C and D of various pH values. A color change was observed in all strips. The intensity of the color of strips dipped in Solution C was slightly reduced after 15 minutes of drying however the color maintained for at least 72 hours. The intensity of the color of strips dipped in Solution D was did not change after drying and for the next 72 hours.

EXAMPLE 7 - Stability of indication II

Product Preparation:

To a 10 ml of Acetone the following ingredients were added: 210 mg Cellulose acetate, 107 μΙ Dibutylphthalate, 32 μΐ Aliquat, 210 μΐ 2-Ethoxy ethanol and 3.2 mg nitrazine yellow dissolved in 210 μΐ DDW. All ingredients were dissolved by stirring the mixture for a few minutes. A polyester monofilament screening fabric was then coated with the polymer solution to give the desired product,

The stability of indication provided by the product of the invention was compared with the stability of indication provided by the following commercial pH indicating articles: nitrazine Paper (Apothecon) and Merck’s pH-Indicator strips for pH ranges of 4.0-7.0. The color changes were compared to a reference PANTONE color guide catalogue and the results are summarized in Table 3. The test solutions which were used in this study consisted of 0.1% BSA in water (double distilled) at pH 5.5. The pH was adjusted with solutions of 1M Hydrochloric Acid or 1M

Sodium Hydroxide solutions.

TABLE 3

<td colspan="4"> Stability of Indication</td>

<td> Time</td><td> Indicator solution</td><td> nitrazine Paper (Apothecon)</td><td> pH Indicator Strip (Merck)</td>

<td> 0</td><td> 120</td><td> 131</td><td> 457</td>

<td> 5 minutes</td><td> 577</td><td> 1245</td><td> 457</td>

<td> 1 hour</td><td> 578</td><td> 117*</td><td> 456</td>

<td> 2 hours</td><td> 578</td><td> 110*</td><td> 456</td>

<td> 24 hours</td><td> 578</td><td> 110*</td><td> 4515*</td>

<td> 48 hours</td><td> 578</td><td> 110*</td><td> 4515*</td>

<td> 72 hours</td><td> 578</td><td> 110*</td><td> 4515*</td>

* indicating incorrect pH values in comparison to manufacturer color scale.

EXAMPLE 8 - Articles for detecting amniotic fluids

Three solutions were prepared:

Solution A: 0.2% Aliquot 336 in DDW (double distilled water);

Solution B: 10 unit/ml urease and 0,003% m-cresol purple in DDW; and Solution C: 0.003% nitrazine yellow in isopropyl ether.

A nitrocellulose membrane was dipped in Solution A and transferred to an atmosphere of 50 mm Hg. After 30 minutes, the membrane was removed from the vacuum, Solution B was applied in a pattern resembling the word NO at a density of 50 μΐ/mm². Solution C was applied in a pattern resembling the word AMNIO at a density of 50 μΙ/mm². The membrane was transferred to an atmosphere of 50 mm

Hg. After 30 minutes, the membrane was removed from the vacuum. The membrane was dipped in a pH 7 test solution. The word AMNIO appeared in purple. After drying at ambient conditions for three hours, no substantial change of color was observed. The membrane was dipped in urine. The word NO appeared in violet.

EXAMPLE 9 Reducing Erroneous Readings of Color-Changing Devices that Give an Indication of Elevated pH in the Vaginal Secretion

The following example discloses the solution to produce an indicator that needs no color-table or scale to read results, that shows the user a stable indication for a few days, and that does not leach even when in contact with liquids for any practical length of time. For the non-invasive continuous monitoring version, the invention discloses a solution to avoid false positive readings due to urine contamination.

The device is a sticker or a pantyliner that contains two different indicator strips, embedded between layers of one-way absorbent tissues. The two indicators have a color transition-point at different pH levels. The color-reactions of the two indicators also have different reversibility in vaginal secretion vs. urine.

The first indicator strip changes color to stable blue, when sensing elevated pH in vaginal secretions (pH strip). The pH strip contains the pH indicator-nitrazineyellow, which has a pKa of 6.6 in aqueous solution, and with the innovative specific composition, changes the color when the vaginal secretion has a pH level of 5.0 or higher (the same innovative specific composition produces indicators for various pH levels, by using other negatively-charged members of the lonizable phenol group).

In a case where vaginal secretion with elevated pH (5.0-7.0) will reach the 10 strips only the pH strip will change color and the change will remain stable for a few days.

Method of Preparation (1) pH Strip:

Step 1: To a 10 ml of Acetone add 150 mg Cellulose acetate, 107 μΐ

Dibutylphthalate, 23 μΐ Aliquat, 150 μΐ 2-Ethoxy ethanol and 2.4 mg nitrazine yellow • dissolved in 150 μΐ DDW.

Step 2: Stir the mixture for few minutes to complete dissolving.

Step 3: Coat a polyester monofilament screening fabric with the polymer solution (coating other materials un-sensitive to acetone will produce various devices for various using instructions, with the same features).

(2) Urine Strip:

First layer-step 1: To a 4.15 mL DDW add 45 mg PVP, 0.325 mL 25 urease/glycerol solution.

First layer-step 2; Coat a polyester monofilament screening fabric with the polymer solution.

First layer-step 3: The coated strips are dried-out over night at room temperature.

Second layer-step 1: To a 10 ml of THF add 150 mg Cellulose acetate, 107 μΐ

Dibutylphthalate, 23 μΐ Aliquat, 150 μΐ 2-Ethoxy ethanol and 1.2 mg m-Cresol purple dissolved in 120 μΐ 1-Propanol.

Second layer-step 2: Stir the mixture for few minutes to complete dissolving Second layer-step 3: Coat the strip with the second polymer solution.

Second layer-step 4: After drying over night the wash the strip in a saline solution.

The device can be in the form of a swab with a tip produced in the same way as mentioned above, under the header: pH strip. The tip may be prepared by using a short strip, rolled on the stick of the swab, or by coating the tip of an integrated swab (implementing step 3), where the tip consists of any screening fabric.

EXAMPLE 10 A Device Able to Distinguish Accurately Between an Amniotic Fluid Leak or an Elevated pH Vaginal Discharged Secretion and Wetness Caused by Urine Incontinence

Due to the severe consequences of amniotic fluid leakage, pregnant women undergo heavy stress and tend to seek for a health care provider upon any wet sensation in the area of the vagina. The common ways to checks for the presence of amniotic fluid are by examining the pH of vaginal secretions with pH indicators such as nitrazine indicators, running the Fern-test or by visually identifying the source of the leakage.

Amniotic fluid has a pH level that varies between 6-8 and can be identified by a purple-blue color of a nitrazine indicator. Since urine, has a pH level that varies between 5.0-8.0, measuring pH levels as a sole criterion can mislead to erroneous decisions. As the other two ways can be performed only in clinics and hospitals, and by trained staff, there is no practical solution for home monitoring.

In some situations, after amniocentesis tests and other occasions such as high30 risk pregnancies, there is a possibility of small amniotic leaks that can be detected only by continuous monitoring.

Current solutions and earlier inventions fail to serve as a home-use continuous monitoring device, as they leach in fluids, the color change is unstable, and the overlap between amniotic fluid pH level and the urine pH level misleads the users in as 30% of the cases.

The overlap of pH levels, between amniotic fluids and urine is also a great disadvantage for physicians treating patients with wet sensations. Providing pregnant women with a home-use continuous monitoring device, that distinguishes amniotic fluid leakage from urine incontinence with no false alarms, enabling the result reading at personal timing and discretion, and detects any small amniotic leak instantaneously, can on one hand help bring the user in-time to hospital when needed, and on the other hand avoid unnecessary hospitalization and concomitant patient stress.

Providing physicians with a reliable clinic instantaneous detecting article, that distinguishes amniotic fluid leakage from urine incontinence with no false alarms, can serve them by far better than available solutions today.

The article can be a sticker or a pantyliner with an embedded indicator strip. The strip contains the pH indicator-nitrazine-yellow which has a pKa of 6.6 in aqueous solution.

Reaction of the indicator with amniotic fluid (pH 6-8) changes the color from yellow to stable dark blue. Reaction of the indicator with urine (pH 5-8) changes the color to fading green or fading turquoise. Urine with lower pH 5-5.5 does not change the indicator color.

The difference between the color reaction of the indicator with amniotic fluid and with urine consists of two parameters: the chemical composition of the fluids and the indicator’s polymer chemical structure. The different chemical reactions are demonstrated by equations 3 and 5, above.

EXAMPLE 11 - A Device Able to Distinguish Accurately Between Normal Urine and Infected Urine

The reoccurrence of urinary tract infections in certain patients present the need 5 to quickly and easily diagnose whether the patient has another urinary tract infection. Presently, to determine if a patient has a urinary tract infection they must make an appointment to visit a doctor. Furthermore, if the patient is susceptible to the reoccurrence of urinary tract infections they must make periodic visits to the doctor's office to ensure that the infection has not reoccurred. Having a device that would allow the user to determine if they had a urinary tract infection again would minimize stress and time consumed by visits to the physician office and result in quicker diagnosis of the infection, resulting in a reduction in pain suffered by the patient and a more timely treatment of the infection.

The article in this example is a diaper or a panty liner with an indicator that can distinguish between normal urine and infected urine. The user wears the article so that urine can come in contact with the article. The reaction of the indicator with urine (pH 5-8) .changes the color from yellow to green or turquoise. The drying process of the indicator strip at room temperature is short (5 minutes). When normal urine comes in contact with the indicator strip the color changes fade while drying. The color change is completely reversible and the strip becomes yellow again. On the other hand when infected urine conies in contact with the indicator strip the color changes to green or turquoise and stay constant while drying.

The reversibility of the color changes depends on two different environmental factors:

<td></td><td> 1.</td><td> Chemical environment:</td>

<td></td><td> (a)</td><td> The pH level of the fluid-pH level higher than the pKa gives a stable color change.</td>

<td> 30</td><td> (b)</td><td> Buffer capacity of the solution explained extensively above.</td>

<td></td><td> (c)</td><td> Ammonium salts content in the solution-explained extensively above.</td>

2'. Biological environment:

(a) Protein presence in urine gives a stable color change and the reaction is not reversible.

Infected urine provides a stable color change to the indicator, which color 5 change is not reversible. Furthermore, bacteria presence in vaginal secretion fluid also gives a stable color change so that the color change is not reversible.

In solution the color change is governed by the pH and the buffer capacity. On the drying process of the polymer matrix, the NY complex dissociates easily when the buffering system is highly concentrated or when there is high concentration of protonated cations like NH/ in urine. The dissociation of the complex occurs due to the basic nature of the phenolate ion in the NY complex and the high concentration of protons compare to the Ion balance reagent. The dissociation of the complex express in color changes from green or blue to yellow.

On the other hand when the buffer is weak or the medium is highly watery the NYcomplex stay stable at any color including the light green one. The stability of the color is due to the lack of protons compared to the Ion balance reagent concentration.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify ‘and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed chemical structures and functions may take a variety of alternative forms without departing from the invention. Thus the expressions means to . . . and means for . . . , or any method step language, as may be found in the specification above and/or in the claims below, followed by a functional statement, are intended to define and cover whatever chemical structure, or whatever function, which may now or in the future exist which carries out the recited function, whether or not precisely equivalent to the embodiment or embodiments disclosed in the specification above, i.e., other means or steps for carrying out the same functions can be used; and it is intended that such expressions be given their broadest interpretation.