FIELD OF THE INVENTION

The present invention is related to a coupon which is particularly suited for use in colorimetric-based chemical analysis of liquids. More particularly, the present invention is related to a coupon design which provides for improved sensitivity and uniformity in colorimetric-based chemical analysis due to an increased optical path length for light transmission through a developed liquid column with minimal structural barriers.

BACKGROUND

Colorimetric-based chemical analysis is a widely used technique for many chemical assays including, biologically relevant analytes, tap water quality testing, industrial and environmental testing, pool and spa testing and the like as set forth in U.S. Pat. No. 6,541,269 which is incorporated herein by reference. Colorimetric based chemical analysis can be used for a variety of analytes including metals, such as copper or iron, inorganics, such as free chlorine or total chlorine, and biologically relevant components.

Indicators useful for evaluating chlorine include benzidine-type chromogens, diaminothiobenzo-phenone-type chromogens, and azine compounds such as syringaldazine and vanillinazine. Indicators useful for evaluating iron include 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ), and indicators useful for evaluating copper include 5-(4-dimethylaminobenzylidene)rhodanine.

Benzidine indicators are especially useful for evaluating free chlorine, including the 3,3′,5,5′-tetraalkylbenzidines, wherein alkyl is C1-C3 alkyl, in particular methyl and ethyl, and 3,3′,5,5′-tetramethyl-benzidine (TMB) is particularly preferred. Other benzidines within the foregoing formula, include 3,3′-dimethyl, 5,5′-diethyl benzidine and 3,3′,5,5′-tetraethylbenzidine. The four alkyl groups may be the same or different. To prevent leaching from the carrier in a water-based sample, the free base form is typically preferred.

Diaminothiobenzophenone indicators are especially useful for evaluating total chlorine, including the 4,4′-bis(dialkylamino)thiobenzophenones, wherein alkyl is C1-C3 alkyl, in particular methyl and ethyl, and 4,4′-bis(dimethylamino)thiobenzophenone is particularly preferred.

Particularly preferred biological samples are described in U.S. Pat. Nos. 5,563,042 and 3,298,789, both of which are incorporated herein by reference, wherein the analysis of blood samples is described. Fresh, whole blood (typically 20-40 μl) is placed on an ethylcellulose-coated reagent pad containing an enzyme system having glucose oxidase and peroxidase activity. The enzyme system reacts with glucose and releases hydrogen peroxide. The pad also contains an indicator which reacts with the hydrogen peroxide in the presence of peroxidase to give a color proportional in intensity to the sample's glucose level.

A particular disease which is difficult to test for is phenylketonuria (PKU) which is a rare metabolic disorder that occurs in the USA at a frequency of about 1 in 10,000 children. PKU is caused by a genetic defect in the liver enzyme phenylalanine hydroxylase which converts the amino acid phenylalanine (Phe) to tyrosine. If untreated, Phe levels in the blood stream rise to toxic levels which can lead to irreversible brain damage and mental retardation within a few months after birth. For this reason, newborns are screened in the United States for the presence of PKU. If diagnosed, treatment involves strict metabolic control using a low-Phe diet combined with special medical foods preferably initiated within the first seven days of life and followed throughout life. If properly controlled, normal health and development can be expected. Current guidelines recommend that blood plasma levels be maintained between 120-360 μM (2.0-6.0 mg/dL) for PKU patients of all ages.

Phe levels are typically monitored by dried-blood-spot samples taken at home and mailed to a participating laboratory for analysis. Clinical recommendations are for blood Phe levels to be checked once per week for the first five years of life and at least one a month thereafter. Women of childbearing age with PKU must be especially careful to maintain low blood Phe levels which is preferably monitored by weekly blood monitoring prior to conception, and throughout pregnancy, or birth defects can result even if the fetus does not have PKU.

Reducing the impact of PKU has been exasperated by the fact that a blood test for Phe typically requires 5 to 10 days for results to be returned to the patient or the patient's family, making it difficult to control blood Phe since any dietary impact may not be determined for 5 to 10 days and, even then, long after any dietary perturbation. The lag-time between sampling and obtaining the results can cause substantial problems in the dietary regulation of blood Phe levels thereby making it very difficult to maintain Phe in the desired level on a daily basis.

In about 1934 it was discovered that the condition of PKU resulted in abnormally high levels of phenylpyruvic acid (PPA) in urine, which is the result of the liver's inability to properly metabolize Phe. This realization eventually led to the development of a colorimetric urine test for PPA in the late 1950s as the first newborn screening test for PKU. The test involved a reaction with ferric chloride and the reaction was used in a standard test utilizing ferric-chloride-impregnated test strips. Though somewhat beneficial, the test had poor sensitivity with a lower range of detection associated with blood Phe levels of about 900 μM. Therefore the test was only suitable for the initial detection of PKU but the test was not suitable for routine monitoring of blood Phe levels once they were brought under dietary control to be within the therapeutically recommended range of between 120-360 μM.

There has been a long-standing desire for improved methods and systems for colorimetric-based measurement of analytes. There is a particular need for a more immediate test for blood Phe levels and preferably an in-home, or point-of-care, Phe-monitoring system to allow patients the opportunity to engage in more time-responsive maintenance of blood Phe levels. Efforts to achieve an at-home blood test have thus far not been successful. A urine based test is provided herein wherein a novel coupon provides for a convenient method of sampling and monitoring of blood Phe levels for individuals with PKU.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an improved method for colorimetric-based chemical analysis.

It is a particular object of the invention to provide a test for analytes in a liquid using colorimetric-based chemical analysis.

It is a particular object of the invention to provide a urine test for blood Phe concentration suitable for diagnosing or monitoring of PKU.

A particular feature of the invention is the ability to provide an at-home, or point-of-care, measurement of PPA and creatinine (CRE) concentration in urine simply and at a low cost. CRE measurement is used as the standard method to normalize urine test results for dilution effects due to the degree of patient hydration.

These and other embodiments, as will be realized, are provided in a system for monitoring an analyte concentration in a liquid sample. The system includes a coupon comprising an absorbent body with a window through the absorbent body wherein the liquid is maintained in the window by a combination of capillary action and liquid surface tension. A reactant is in the absorbent body wherein the reactant, and/or reaction products caused by the reactant, are capable of diffusing into the window to react with an analyte in the test liquid, or reacted analyte from the absorbent body itself is capable of mixing with the analyte liquid in the window by diffusion or convection, wherein the analyte is present in an analyte concentration, to form a reactant with a color wherein the color has an intensity that correlates to the analyte concentration. A light source is provided which is capable of passing a light beam into the window wherein the light is attenuated by the color proportional to the analyte concentration to form attenuated light. A detector is provided which is capable of measuring an intensity of the attenuated light. Alternatively, the color response of the coupon can simply be viewed by eye under ambient lighting conditions, with the color response compared to a color chart indicating the relationship between the color and the concentration of the designated analyte.

Yet another embodiment is provided in a method for monitoring an analyte concentration in liquid comprising:

providing a coupon comprising an absorbent body with a window through the absorbent body;

placing the test liquid in the window;

allowing the reactant in the absorbent body to diffuse into the window to react with an analyte in the test liquid, or reacted analyte from the absorbent body itself, to form a color wherein the color has an intensity which correlates to the analyte concentration; passing a light beam into the window wherein the light is attenuated by the color proportional to the analyte concentration to form attenuated light; and
measuring an intensity of the attenuated light. Alternatively, the color response of the coupon can simply be viewed by eye under ambient lighting conditions, with the color response compared to a color chart indicating the relationship between the color and the concentration of the designated analyte.

Yet another embodiment is provided in a system for controlling phenylketonuria. The system includes a coupon comprising an absorbent body with a window through the absorbent body. A reactant is in the absorbent body wherein the reactant, and/or reaction products caused by the reactant, are capable of diffusing into the window to react with PPA or CRE in urine contained in the window, or reacted PPA or CRE from the absorbent body itself is capable of mixing with the liquid in the window by diffusion or convection, to form a color wherein the color has an intensity which correlates to the phenylpyruvic acid or creatinine concentration. A light source is provided which is capable of passing a light beam into the window wherein the light is attenuated by the color proportional to the phenylpyruvic acid or creatinine concentration to form attenuated light. A detector is provided which is capable of measuring an intensity of attenuated light. Alternatively, the color response of the coupon can simply be viewed by eye under ambient lighting conditions, with the color response compared to a color chart indicating the relationship between the color and the concentration of the designated analyte.

