PT90386B - A PROCESS AND DEVICE FOR THE COLORIMETRIC DETERMINATION OF CHEMICAL AND BIOCHEMICAL COMPOUNDS (ANALYSIS) IN AQUEOUS FLUIDS - Google Patents

Abstract

A method for determining the presence of an analyte in a fluid is described along with various components of an apparatus specifically designed to carry out the method.

Description

Description referring to LifeScan, Inc., North American, industrial and commercial patent, established at 2443 Wayandotte Street, Mountain View, CA 94043—2312, United States of America, (inventors: Roger Phillips, Geoffery McGar raugh, Franklin A. Jurik and Ray mond D. Underwood, residing in the USA), for ⁿ PROCESS AND DEVICE FOR THE COLORIMETRIC DETERMINATION OF CHEMICAL AND BIOCHEMICAL COMPOUNDS (ANALYTICAL) IN WATER FLUIDS ».

DESCRIPTION scope of the invention

The present invention relates to a test method and device for colorimetric determination of chemical and biochemical (analytical) components in aqueous fluids, especially whole blood. In a preferred embodiment, it relates to a test method and device for measuring the concentration of glucose in whole blood by means of colorimetry.

Background of the invention

It is of increasing importance to quanti—

N.

fication of chemical and biochemical components in aqueous colored fluids, especially colored biological fluids such as whole blood and urine and derivatives of biological fluids such as blood serum and blood plasma. There are important applications in the diagnosis and medical treatment and when 4 * The tification of exposure to therapeutic drugs, intoxicants, dangerous chemicals and the like. Sometimes the quantities of materials to be determined are either very small - in the range of one microgram or less per deciliter - or so difficult to determine with precision that the device used

Iized is complicated and useful only for specialized laboratory personnel. In this case, results are generally not available within a few hours or days after sampling. At other times, there is usually an emphasis on the ability of lay operators to perform the test routinely, quickly and reproducibly outside of a laboratory environment with rapid

Μ M or immediate presentation of information.

A common medical test or analysis is the measurement of blood glucose levels by diabetics. Current teachings advise diabetic patients to measure their blood glucose level from two to seven times a day depending on the severity of the disease or their individual cases. Based on the model observed in the measured glucose levels, the patient and the doctor together make adjustments to the diet, exercise and injection of insulin to better control the disease. Of course, this information must be immediately available to the client.

Currently, a drop method used in the United States employs a test article of the type described in United States Patent No. 3,298,789 issued on January 17, 1967 to Mast. In this method, a sample of fresh, whole blood (usually 20—40 µl) is placed on a reagent pad coated with ethyl cellulose that contains 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 that reacts with hydrogen peroxide in the presence of peroxidase to provide a color proportional in intensity to the glucose level of the sample.

Another method of testing blood glucose employs similar chemistry but instead of the ethyl-coated pad — cellulose employs a water-resistant film through which the enzymes and indicator are dispersed. This type of system is described in United States Patent 363O 957 issued on December 28, 1971 to Rey et al.

In both cases, the sample is allowed to remain in contact with the reagent pad for a certain time (usually one minute). Then, in the first case, the blood sample is washed with a stream of water while in the second case the film is cleaned. The reagent pad or film is then wiped dry and evaluated. The evaluation is made either by comparing the color obtained with a color table or by placing the pad or film on a reflectance instrument to read a light intensity value.

Although the methods mentioned above for controlling glucose have been used for years, they have certain limitations. The sample size required is quite large for a finger prick test and is difficult to obtain for some people whose capillary blood is not immediately felt.

In addition, these methods share a limitation— ** ** # with other simple colorimetric determinations for lay operators in which their result is based on an absolute color reading which is in turn related to the absolute extent of the reaction between the sample and test reagents. The fact that the sample must be washed or cleaned from the reagent pad after the reaction time interval requires the user to be ready at the end of the time interval and to clean or apply a wash flow in due course. the reaction stopping by removing the sample leads to some uncertainty in the result, especially in the hands of the home user. Excessive washing may have low results and insufficient washing may result from high results.

Another problem that often exists in simple colorimetric determinations for lay operators is the need to start a time sequence when applying blood to a reagent pad. A user will normally cause a finger prick to obtain a blood sample and will then need to »simultaneously» apply the finger finger to a reagent pad while starting a time loop with his other hand »requiring the two hands simultaneously. This is particularly difficult since it is often necessary to ensure that the time loop starts only when blood has been applied to the reagent pad. All prior art methods nec

M ** requires additional manipulation or additional circuitry to obtain this result. Thus, it is desirable to simplify this characteristic of reading instruments by reflection.

The presence of red blood cells or other colored components often interferes with the measurements of these absolute values, thus requiring the exclusion of red blood cells in these two previous methods as has been widely practiced. In the North America Patent device at No. 3,298,789, an ethyl cellulose membrane prevents red blood cells from entering the reagent pad. Likewise, the waterproof film of U.S. Patent No. 3,630,957 prevents red blood cells from entering the cushion. In both cases, washing or cleaning also acts to remove these red blood cells that potentially interfere, before the measurement.

- 4 Thus, there remains a need for an analyte detection system in colored liquids, such as blood, which does not require the removal of excess liquid from a reflectance strip from which a reflectance reading is obtained.

Summary of the invention

New methods are provided) compositions and devices for diagnostic tests that comprise a porous hydrophilic matrix containing a signal production system and a reflectance measurement device that _ ** A is activated by a change in the reflectance of the matrix when the fluid penetrates the matrix. The method comprises adding the sample (normally whole blood) to the matrix which filters out large particles, such as red blood cells, usually with the matrix present in the device. The signal production system produces a product that subsequently alters the reflectance of the matrix, the alteration of which can be related to the presence of an analyte in the sample.

An example of the diagnostic test system is the determination of glucose in whole blood, in which the determination - ** Λ mining is done without interference from blood and without a complicated protocol subject to error.

• v

Brief description of the drawings

The present invention can be better understood with reference to the following detailed description when read in conjunction with the accompanying drawings, where. *

Figure 1 is a perspective view of a ** realization of a test device containing the reaction pad ** of the reaction to which the fluid to be analyzed is applied)

Figure 2 is a schematic diagram of a

- 5 device that can be used in the practice of the present invention »

Figure 3 is a perspective view of a preferred embodiment of the test device of the present invention— “w ** vention placed within a measurement system *

Figure 4 is an enlarged plan view of a preferred embodiment of the test device of the present invention placed within a measurement system *

Figure 5 δ is a graph showing a second order color to eliminate errors due to effects

Μ M of chromatography when using the present invention *

Figures 6a and 6b are scanning diagrams of glucose values as measured by a preferred embodiment of the present invention (called the single wave length MPX system) graphically represented as opposed to the glucose values of Iellow Springs Instruments (YSIj and

Figures 7a * 7b »7c and 7d are scanning diagrams of glucose values as measured by a second preferred embodiment of the present invention (called dual wavelength MPX system) graphically represented in opposition to the Yellow Springs glucose values Instriments ”(YSI).

Detailed description of the reagent element invention

The present invention provides a fast and simple improved methodology that uses a safe and easy to operate device for the determination of analytes such as glucose * specifically involving an enzyme substrate that results in the production of hydrogen peroxide as a pro

- 6 enzyme duct. The method includes applying to a porous matrix a small volume of whole blood sufficient to saturate the matrix. Note that the present system is capable of determining glucose levels from optical readings of whole blood samples. It is unnecessary to remove the blood plasma from the sample and the present invention avoids the need for this step. In addition »this system is - ** capable of making correct readings as long as only a small volume saturates the test strip matrix. Beyond this limit, the reading is independent of the volume. Connected to the matrix are one or more reagents from a signal production system 'which results in the production of a product resulting in an initial change in the reflectance amount of the matrix. The matrix is normally present ** A in a reflectance measurement device when blood is applied. The liquid sample penetrates the matrix, resulting in an initial change in reflectance on the measurement surface. The reading is then made one or more times after the initial change

Λ ** ciai of reflectance to refer to the subsequent change in the reflectance on the measuring surface or in the matrix as result «tf« tf of the formation of the reaction product the amount of analyte in the sample.

For blood measurements »especially glucose measurements» whole blood is normally used as the test vehicle. The matrix contains an oxidase enzyme that produces hydrogen peroxide. Also contained in the matrix will be a second enzyme »specifically a peroxidase» and a dye system which produces a light absorbing product together with the peroxidase. The light absorption product changes the reflectance signal of the matrix system. With whole blood »the readings are made in two different wavelengths» with the reading of a wavelength used to separate the background interference caused by the matocrit »blood oxygenation» and other variables that can affect the result. Thus, the present invention is capable of analyzing whole blood samples.

- 7 A reagent element is used that includes the matrix and the members of the signal production system contained in the matrix. The reagent element can include other components for specific applications. The method requires the application of a small volume of blood, which has not normally been subjected to previous treatment (unless optionally treated with an anticoagulant)> to the matrix. The measurement time period is activated or initiated * by the device by detecting au · * Atopically a change in the reflectance of the matrix when ** A fluid enters the matrix. The change in reflectance beyond a predetermined time period as a result of the formation of the reaction product is then related to the amount of analyte in the sample. The intensity of the light source used to analyze the sample is, of course, »also carefully controlled and regulated to ensure that the measurement can be repeated.

The first component of the present invention to consider is a reagent element, conveniently in the form of a pillow, which includes an inert porous matrix and the component or components of a signal production system »whose system is susceptible to reaction with an analyte to produce «4 4 * produce a reaction product of light absorption, impregnated in the« 4 4 * pores of the porous matrix. The signal production system does not significantly imply the flow of the liquid through the matrix.

In order to assist the reading of the reflectance, it is preferable that the matrix has at least one side that is smooth and flat. Normally, the matrix is formed on a thin sheet with at least one smooth and flat side. In use, the sample of liquid to be analyzed is applied to one side of the sheet in which any test compound present passes through the reagent element by means of capillarity, twisting, gravity flow or diffusion. The components of the «4« 4 signal production system present in the matrix will react to give ** 4W a light absorbing reaction product. The incident light

finds the reagent element in a different location than the one to which the sample is applied. The light is then reflected from the element's surface as diffused reflected light. This diffused light is received and measured, for example by the detector of a reflectance spectrophotometer. The amount of reflected light will be related to the amount of analyte in the sample, normally being an inverse function of the amount of analyte in the sample.

The matrix

Each of the components necessary for the ** # production of the reagent element will be described in time. The first component and the matrix itself.

The matrix will be a porous hydrophilic matrix to which the reagents are covalently or non-covalently attached. The matrix will allow an aqueous vehicle to flow through the matrix. It will also allow the binding of protein compositions to the matrix without significantly adversely affecting the biological activity of the protein, i.e., the enzymatic activity of an enzyme. To the extent that proteins must be covalently linked, the matrix will have sites for covalent binding or can be activated by means known in the art. The matrix composition should be reflected and of sufficient thickness to allow the formation of a light absorbing dye ** in the unoccupied volume or on the surface in order to substantially affect the reflectance from the matrix. The matrix can be of a uniform composition or a coating on a substrate that provides the necessary physical properties and structure.

The matrix will not deform when wet, thus retaining its original shape and size. The matrix will have a defined absorption, so that the volume that is absorbed can be calibrated within reasonable limits, with variations normally kept below about 5θ% »preferably

- 9 no more than 10%. The matrix will have sufficient water resistance to permit routine manufacture. The matrix will allow non-covalently bound reagents to be distributed relatively evenly over the surface of the matrix.

Examples of matrix surfaces are polyamides, especially with samples that involve whole blood. Polyamides are condensation polymers of monomers of between 4 and 8 carbon atoms, where the monomers are lactams or combinations of diamines and di-carboxylic acids. Other polymer compositions that have comparable properties can also be used. Polyamide compositions can be modified to introduce other functional groups that provide loading structures, so that the matrix surfaces can be neutral, positive or negative, as well as neutral, basic or acidic. Preferred surfaces are positively charged. It was concluded that this positive charge increases both stability and durability.

When used with whole blood, the porous matrix preferably has pores with an average diameter comprised between 0.1 and 2.0 µm, preferably between 0.6 and 1.0 pm. When the porous matrix contains pores having an average diameter of 0.8 µm, the blood sample will not cause a chromatographic effect. This means that the blood sample will not attempt to leave the edges of the circular matrix. On the contrary, the blood remains located within all pores of the matrix and provides a possibility of uniform reading of the entire matrix. In addition, this pore size maximizes the non-staining effect of blood. That is, the size of the well is simultaneously adequately filled, but not overfilled, so that the blood hematocrit level is not the cause of the sample needing to be dried before reading the sample. It was also concluded that pores of this size are optimal when considering durability and stability.

A preferred way of preparing the porous material is to mold the hydrophilic polymer into a core of non-woven fibers. The core fibers may be of any fibrous material that has the described integrity and strength, such as polyesters and polyamides. The reagent that will form the light absorbing reaction product, which will be presented in detail later, is present within the matrix pores but does not block the matrix so that the liquid portion of the test vehicle, for example blood, when being analyzed, flow through the pores of the matrix, while particles such as erythrocytes are retained on the surface.

The matrix is substantially reflective to provide diffuse reflectance without the use of a reflective support. Preferably at least 25%, more preferably at least 5θ%> of the incident light applied to the matrix is reflected and emitted as diffuse reflectance. A matrix with a thickness of less than 0.5 mm is normally used, with a thickness between 0.01 mm and 0.3 mm being preferred. Most preferably, a thickness between 0.1 mm and 0.2 mm, specifically for nylon matrix.

Normally, the matrix will be connected to a support in order to provide you with physical form and rigidity, although this may not be necessary. Figure 1 shows an embodiment of the present invention in which there is a strip 10 that has a thin pad of hydrophilic matrix 11 placed on one end of a plastic support or handle 12 by means of an adhesive 13 that firmly and directly joins the reagent pad 11 the handle 12. There is a hole 14 in the plastic holder 12 at the location to which the reagent pad 11 is attached so that the sample can be applied to one side of the reagent pad and the reflected light from on the other side.

A sample of liquid to be tested is applied to pad 11.

Generally, with blood as a specimen of a sample to be tested, the reagent pad will be in the order of 10 nm | 2 to 100 mm ^ area, especially 10 mm ^ to 5θ mm ^ area (or having a diameter of about 2 mm to 10 mm), which normally has a volume that will be more than saturated with 5 — Ιθ microliters of sample. Of course, once saturation above the 5—10 microliter limits is reached, no further blood is required.

# **

Diffuse reflectance measurements were performed in the prior art using a reflective support attached or placed behind the matrix. Such support is not necessary or will normally be present during the practice of the present invention, either as part of the reagent element or the reflectance device.

As can be seen in figure 1, the holder is secured to the reagent pad 11 so that a sample can be applied to one side of the reagent pad 11 while measuring the reflectance of light from the other side of the opposite reagent pad 11 to the place where the sample is applied ·

Figure 2 represents a system in which the reagent is applied to the side with hole 14 in the handle of the support 12 while the light is reflected and measured on the other side of the reagent pad 11. Other structures may be used in addition to that described . The pad 11 can take various forms and is% · * matted, subject to the limitations included here. Pad 11 must be accessible at least on one surface and usually on both.

The hydrophilic layer (reagent element) can be attached to the support by any convenient means, for example, a support, staple or adhesives, however, in the preferred method and attached to the base. Bonding can be done with any non-reactive adhesive, by a thermal method in which the base surface is sufficiently fused to hold some of the material used for the hydrophilic layer or by ultrasonic or microwave bonding methods that they also connect the hydrophilic pads of the sample to the base. It is important that the connection is such that it does not interfere in any way ** A substantial with diffuse reflectance measurements or with the reaction to be measured, although this is unlikely to occur as no adhesive is required where it is made. the reading. For example, an adhesive 13 can be applied to the base strip 12 followed first by drilling the hole 14-in the combined strip and adhesive 'tf and then apply the reagent pad 11 to the adhesive near the hole l4 so that the peripheral portion of the reagent pad attaches to the base strip.

Chemical reagents

Any signal production system that is capable of reacting with the sample analyte can be used to produce (either directly or indirectly) a compound that is characteristically absorptive at a wavelength different from the wavelength at which the vehicle test absorbs substantially.

«Tf

Polyamide matrices are particularly easy to carry out reactions in which the substrate (analyte) reacts with an oxidase enzyme that uses oxygen in such a way as to produce a product which subsequently reacts with a daily dye intermediary to form a direct or indirect dye which absorbs at a predetermined wavelength range · For example, an oxidase enzyme can oxidize a substrate and produce hydrogen peroxide as a reaction product. The oxygen peroxide can then be reacted with a dye intermediate or precursor, in a catalysis or non-catalysis reaction, to produce an oxidized form of the intermediate or precursor. This oxidized material can produce the colored product or react with a second precursor to form the final dye.

Non-limiting examples of typical analyzes and reagents include the following materials in the list below

Type of sample and analyte Reagents

Glucose in blood? serum? urine or other biological fluids? wine? fruit juices or other colored aqueous fluids. Is full blood a particularly preferred type of sample? because separation is time-consuming and impractical with domestic use.

Glucose oxidase? Peroxidase and an oxygen acceptor.

Oxygen acceptors include:

O — dianizine (l)

0 — toluidine

0 — tolidine (l)

Benzidine (l)? 2 ’—azinodi— (3-ethyl — benz — thia— zolin sulfonic acid (6)) (1)

3-methyl-2 — benzothiazolinone hi— drazone and N? N — dimethylaniline (1) Phenyl and 4 — aminophenazone (1)

2? 4 — sulfonated dichlorophenol and

4-aminophenazone (2)

3— methyl — 2 — benzothiazolinone hydra zona and 3- (dimethylamino) benzoic acid (3)

2 — methoxy — 4 — allyl phenol (4)

4— amino — antipyrene — dimethylaniine (5) (1) As described by Clinicai Chemistry? Richterich and Columbus? P. 367 and references cited here (2) Analyst? 97? (1972) 142-5 (3) Anal. Biochem. 105 (1980) 389-397 (4) Anal. Biochem.? 79? (1977) 597-601 (5) Clinica Chemica Acta? 75? (1977) 307—391

All included here for reference.

Analysis Method analysis method for the present invention is based on a change in absorption, as measured by diffuse reflectance, which is dependent on the amount of analyte present in the sample to be tested. This change can be

M ** ** determined by measuring the change in the absorption of the test sample between two or more points in time.

first step of the test to be considered will be the application of the sample to the matrix. In practice, an analysis can be carried out as follows:

First, a sample of aqueous fluid containing an analyte is obtained. Blood can be obtained by pricking the finger, for example. An excess of this fluid is applied, beyond the limits

- Λ of saturation of the matrix in the area where the reflectance will be measured (i.e., about 5 ~ 1θ microliters) to the test element reagent elements. It is not necessary to simultaneously start a chronometer (as is commonly necessary in the prior art), as will become clear below, due to the initiation process practiced by the present invention. Can you remove it? —If the excess fluid, such as drying in the light, but such removal is also not necessary. The test device is normally mounted on an instrument for reading light absorption, for example, color intensity by reflectance, before applying the sample. The absorption is measured at certain points in time after the application of the sample. The absorption concerns in this application not only the light within the visual limit with the wavelength but also outside the visual limit of the wavelength, such as infrared or ultraviolet radiation. From these absorption measurements, the speed of color development can be calibrated in terms of analyte level.

Measuring instrument

An appropriate instrument can be obtained,

as a diffuse reflection spectrophotometer with appropriate software, to automatically read the reflection at certain points in time, calculate the rate of change of the reflectance and, using calibration factors, control the level of the analyte in the aqueous fluid. Such a device is schematically represented in figure 2, in which a test device of the present invention is presented comprising the base 12 to which the reagent pad 11 is attached. The light source 5 '', for example a light emitting diode high intensity (LED) projects a beam of light onto the reagent pad. A substantial portion of this light (at least 25% »preferably at least 35%» and most preferably at least 5θ% »

Λ **. Λ in the absence of the reaction product) and reflected diffusely from the reagent pad and is detected by the light detector 6, for example a phototransistor that produces an output current proportional to the light it receives.

If desired, the light source 5 and / or the detector 6 can be adapted to generate or respond to a light of specific wavelength. The output product of detector 6 passes to amplifier 7> for example, a linear integrated circuit that converts the phototransistor current into voltage. The amplifier product 7 can be distributed to the tracking and detection circuit 8. This is an integrated linear / digital combination circuit that locates or follows the analog voltage from amplifier 7 and, under the control of microprocessor 20, blocks or keep the voltage level at that time.

The analog / digital converter 19 receives the analog voltage from the tracking and detection circuit 8 and converts it, for example, into a twelve-bit digital binary number under the control of microprocessor 20. The microprocessor 20 can be a binary integrated circuit. It serves the following control functions: 1) time regulation for the entire system} 2) reading of the output product of the analog / digital converter 19i 3) together with the program and the data memory 21, stores

data corresponding to the reflectance measured at certain time intervals) 4) calculation of the analytical levels from the stored reflectances) and 5) output of the analyte concentration data output to the display 22. Memory 22 can be a digital integrated circuit that stores the data and the microprocessor operating program. Said device 22 can take various forms. It is usually a visual display, such as a liquid crystal display (LCD) or LED, but it can also be a ribbon printer, an audible signal, or the like. The instrument can also include a start / stop switch and can provide a visible or audible time control to indicate times for applying samples, taking readings, etc., if desired.

Reflectance Connection

In the present invention, the reflectance circuit itself can be used to initiate time regulation by measuring a drop in reflectance that occurs when

M «V ** \ the aqueous portion of the suspension solution applied to the reagent pad (eg blood) migrates to the surface on which the reflectance will be measured · Normally, the measuring device is switched to Ready mode in which reflectance readings are automatically taken at very close space intervals (usually about 0.2 seconds) from a strip of non-reactivated, substantially dry reagent, other than white. The initial measurement is usually done before the matrix penetrates the fluid to be analyzed but can be done after applying the fluid to a location on the reagent element other than the location where the reflectance is being measured. The reflectance value is evaluated by the microprocessor, usually by storing successive values in memory and then by comparing each value with the non-reactivated value. When the aqueous solution penetrates the reagent matrix, the reflectance drop signals the beginning of the measurement time interval. Droplets in 5-5θ% reflectance can be used to start timing, usually a drop of about 10%. In this simple mode, there is an exact synchronization of the test vehicle that reaches the surface from which measurements are taken and the sequence of readings is initiated, without the need for user activity.

Although all the systems described in this application are especially directed to the use of polyamide matrices and particularly to the use of such matrices in determining the concentration of various sugars, such as glucose, and other materials of biological origin, it is not necessary limit the binding feature of the reflectance of the present invention to such matrices. For example, the matrix used with the reflectance bond can be formed from any water insoluble hydrophilic material and any other type of reflectance test.

Specific Application to Glucose Test

A specific example related to the detection of glucose in the presence of red blood cells will now be presented in order to highlight the specific advantage and greater detail. Although this represents the preferred embodiment of the present invention, the invention is not limited to the detection of blood glucose.

The use of polyamide surfaces to form the reagent element provides a number of desirable features in the present invention. These include: the reagent element is hydrophilic (ie absorbs the reagent and sample immediately), does not deform when wet (to provide a flat surface for reading reflectance), is compatible with enzymes (in order to provide good stability in duration), absorbs a limited sample volume per unit volume of the membrane (necessary to demonstrate an extensive dynamic range of measurements), and is resistant to—

enough water to allow routine manufacture.

In a characteristic configuration, the present method is carried out using a device consisting of a plastic support and the reagent element (the matrix having imprinted the signal production system itself). The preferred matrix for use in preparing the reagent element is a microfiltration nylon membrane, particularly membranes made of nylon — 66 molded in a core of non-woven polyester fibers. Numerous microfiltration nylon membranes of this type are commercially produced by Pall Ultrapine Filtration Corporation, having pore sizes between 0.1 and 3 µm microns. These materials have mechanical resistance and flexibility, dimensional stability after exposure to water and rapid wetting.

Many variations in the specific chemical structure of nylon are possible. These include nylon-66 not — worked with terminal charge groups (sold under the registered trademark ULTRAPORE of Pall Ultrafine Filtration Corporation, • 'ΡβΙΙ' ¹ ). Positive charges predominate below pH 6 while negative charges predominate above pH 6. In other membranes nylon is worked before forming the membrane to provide membranes with different properties. Nylons worked with carboxy groups are negatively charged in a wide range of pH (sold as CARBOXYDYNE by Pall). Nylons can also be worked with a high density of positive charge groups on their surface, usually groups of quaternary amines, so that there is little variation in charge over a wide pH range (sold as POSIDYNE by Pall). Such materials are particular

- · * Suitable for the practice of the present invention.

It was concluded that keeping the solution pH below 4.8 will help to stabilize the enzymes in the solution. The most efficient level of stability was found at pH 4.0. This results in a storage time at room temperature of 12—18 months. Thus, a positively charged ion strip is desirable.

It is also possible to use membranes that have reactive functional groups called covalent protein immobilization (sold as BIODYNE IMMUNO AFFINITY membranes by Pall). Such materials can be used to covalently bind proteins, for example enzymes, used as reagents. Although all of these materials are usable, nylon having a high density of positive charge groups on its surface provides the best reagent stability when formulated in a dry reagent pad. Un-worked nylons provide the following best stability with the following carboxylated nylons.

Desirable results can be obtained with pore sizes ranging between 0.2 and 2.0 pm, preferably between 0.5 and 1.2 pm, and more preferably about 0.8 pm, when used with whole blood.

The shape of the handle on which the reagent element is mounted is relatively unimportant since the handle allows access to one side of the reagent element by the sample and the other side of the reagent element by the incident light whose reflection is to be measured. The handle also assists in the insertion of the reagent element in the absorption measurement device so that it registers with the epic system. An example of a suitable handle is a Mylar film or other plastic strip to which a transfer adhesive such as 3M 465 or Y946O transfer adhesives has been applied. A hole is made in the plastic through the transfer adhesive. A reagent element, usually in the form of a thin pad, either containing reagents or to which reagents are subsequently added, is then applied to the handle by means of the transfer adhesive so that it is firmly attached to the handle in the area surrounding the hole that was done through the handle and the transfer adhesive.

This device is illustrated in figure 1, which has a strip 10 which has a reagent pad 11 attached to a Mylar film handle 12 by means of adhesive 13 · θ hole 14 allows access of the sample or the light in— «* on one side of the reagent pad 11 although access to the other side of the reagent pad is not prevented. All dimensions of the reagent pad and handle can be chosen so that the reagent pad fits securely on a reflectance reading instrument in a location close to a light source and a reflected light detector. The dimensions of the hole generally range between 2—10 mm in diameter and those of the handle width about 15 mm. A 5 mm diameter hole 14 in the reagent strip shown in Figure 1, works quite satisfactorily. Naturally, there is no specific limit for the minimum diameter of this hole, although diameters of at least 2 mm are preferred for ease of manufacture, application of the sample and reading of the reflection of light.

As can also be seen in figures 3 and 4, strip 10 can be optimally guided in a slot 50 of an image scanning machine 60. This is done by placing a slot 15 on strip 10 near the midpoint of the upper part of the image. strip 10. Having done this, strip 10, when conducted through the sides 55 of the 5θ> slot, will reach the same place repeatedly, to ensure high accuracy in the test results. Such repetitive character is ensured by moving the slot 15 against the pin 65. The strip 10 will rotate around the pin 65 in the slot 15 so that the edges 16 of the strip adapt to the sides 55 of the slot 50. This, of course, also aligns repetitive way the hole l4 above the center of test 80 which includes multiple LEDs 5 in the image scanning machine. 60. This ensures that hole 14 containing the blood sample will have a uniform dose of incident light for analysis.

Although many dyes can be used as indicators, the knowledge will depend on the nature of the sample.

It is necessary to select a dye that has an absorption at a wavelength different from the wavelength at which the red blood cells absorb the light, with whole blood as the test vehicle or other contaminants in the solution to be analyzed with other test vehicles. The pair of MBTH-DMAB dye (methyl 3-methyl-2-benzotiazoIinona hydrazone and 3- "di ^me tilaminobenzóico), although being described above as suitable for color development for peroxidase markers in immunoassays enzymes were never used in a glucose measuring reagent.

** Λ

This pair of dyes provides greater dynamic variation and has improved enzyme stability when compared to traditional dyes used for glucose measurement, such as benzidine derivatives. This enzyme stabilizer makes the MBTH-DMAB dye pair especially desirable to ensure longer test strip durability. In addition, the MBTH-DMAB dye pair is not carcinogenic, a characteristic of most benzidine derivatives. Another pair of dyes that can be used to measure glucose is the pair AAP — CTA (4 — amino — antipyrene and chromotropic acid). Although this pair does not provide as wide a dynamic range as MBTH-DMAB, it is stable and suitable for use in the practice of the present invention, when glucose is given. Again, the AAP — CTA dye pair provides a wide dynamic range and greater stability in enzymatic activity than the more widely used benzidine dyes.

The use of the MBTH-DMAB pair allows the correction of the hematocrit and the degree of oxygenation of the blood with a simple correction factor. The benzidine dyes most commonly used do not allow such a correction. The MBTH-DMAB dye forms a chromophor that absorbs at approximately 635 nm but not to a significant extent at 700 nm. Slight variations in wavelength measurements are allowed. At 700 nm, both the hematocrit and the degree of oxygenation can be measured by measuring the color of the blood. In addition, the diodes

- 22 light emission (LED) are commercially available for the two measurements 635 nm and 700 nm, thus simplifying the mass production of a device. By using the preferred pore size of the membrane described above and the formulation of the reagent, the behavior of both the hematocrit and oxygenation can be corrected by measuring the simple on-line length of 700 nm.

Two additional conditions have been discovered to provide specific stability and long life for a glucose oxidase / peroxidase formulation in a polyamide matrix. Storage is improved at a pH of between 3.8 and 5 ”, preferably between 3, 8 and 4.3, most preferably about 4.0. Likewise »good storage and unexpected stability was found with a mixture of a capping system concentrated to the reagents found in the matrix. It was concluded that the most effective buffer was a 10% by weight citrate buffer, with effective concentrations between 5-15%. These are weight / volume percentages of the solution in which the reagents are applied to the matrix. Other buffers can be used on the same molar base. The greatest stability was achieved using a low pH, preferably pH 4, with a MBTH-DMAB coloring system, and a high enzyme concentration of approximately 500—1000 M / ml of application solution. As previously indicated, these strips prepared using these parameters result in a durability of 12 to 18 months.

When preparing the MBTH-DMAB reagent and the enzyme system that forms the residue of the signal production system, it is not necessary to maintain exact volumes and proportions although the values suggested below show good results. The reagents are immediately absorbed by the matrix pad when glucose oxidase is present in a solution of about 27—54% by volume »peroxidase is present in a concentration of about 2.7-5.4 mg / ml, MBTH it is present in a concentration of about 4—8 mg / ml, and DMAB is present in a concentration of about 8—16 mg / ml. The proportion of the weight

DMAB — MBTH is preferably kept within the range of 1: 1 to 4: 1, preferably from 1.5 * 1 to 2.5 ^J 1 and most preferably from about 2: 1 ·

Basic manufacturing techniques for the reagent element are, once established, straightforward. The membrane itself is strong and stable, especially when selecting a nylon membrane of the preferred embodiment · Only two solutions are required for reagent application, and these solutions are both stable and formulated immediately. The first usually contains the coloring components and the second usually contains the enzymes. When using the HBTH-DMAB dye pair, for example, the individual dyes are dissolved in an aqueous organic solvent, usually in a mixture of acetonitrile i: i and water. The matrix is dipped in the solution, the excess liquid removed by wiping, and the matrix is subsequently dried, usually at 50 ° C - 60 ° C for 10—20 minutes. The matrix containing the dyes is then immersed in an aqueous solution containing the enzymes. The typical formulation contains the enzymes of peroxidase and glucose oxidase as well as any buffer, preservative, stabilizer, or the like. The matrix is then dried to remove excess liquid and dried as before. A standard formulation for the glucose reagent is as follows:

Dye bath

Picture:

40 mg in MBTH 8th mg in DMAB 5 ml in acetonitrile, and 5 ml in Water

Stir until all the solids are dissolved and pour into a glass dish or other flat surface. A Posidyne membrane (Pall Co.) is immersed, the excess liquid is dried, and dried at 5 ° C for 15 minutes.

Enzyme bath

Picture:

6 ml in Water 10 mg in EDTA, salt di-sodium, 200 mg in Sigma Poly Pep, low viscosity 0.668 g in citrate in only hate

Citric acid,

0.523 g of

2.0 ml in of 30 mg in 3.0 ml in

TM by weight of GAF Gantrez AN — 139 dissolved— in horseradish peroxidase water, 100 units / mg, and glucose oxidase, 2000 units / ml

Shake until all solids are dissolved and pour into a glass dish or other flat surface. A membrane previously impregnated with dyes is immersed, the excess liquid is dried, and dried at 56 ° C for 15 minutes.

The electronic device used to take the reflectance readings contains, at a minimum, a light source, a reflected light detector, an amplifier, an analog / digital converter, a microprocessor with memory and program, and a display device, as shown in figure 2.

The light source usually consists of a light emitting diode (LED). Although it is possible to use a polychromatic light source and a light detector capable of measuring at two different wavelengths, a preferred apparatus should contain two LED sources or an optical diode capable of emitting two different wavelengths of light. Commercially available LEDs that produce light wavelengths described as being preferred in this description include a Hewlett Packard HLMP — 1340 with a maximum emission at 635 nm and a Hewlett Packard QEMT — 1045 with a maximum narrow band emission at 700 nm. Commercially available light detectors include a Hammamatsu hl «Ltí *»

5074 — l8K and a Litronix BPX — 65.

While other methods of making measurements are possible, the following method has provided the desired results. Readings were made by means of the photodetector at determined intervals after the timing started. The 635 nm LED is activated only for a short period of measurement time starting approximately 20 seconds after the start-up time as indicated by the reflectance connection system described above. If this reading indicates that a high level of glucose is present in the sample, a reading of 3θ seconds is taken and used in the final calculation to improve accuracy. Normally, high levels are considered to start at around 250 mg / dl. The antecedent is corrected with a reading at 7θ0 nm taken about 15 seconds after the beginning of the measurement period. The reading from the photodetector is integrated into the interval while the appropriate LED is activated, which usually happens in less than a second. The non-worked reflectance readings are later used for calculations performed by the microprocessor after the signal has been amplified and converted into a digital signal. Numerous microprocessors can be used to perform the calculation, an AIM65 single keyboard microcomputer manufactured by Rockwell Internatio nal proved to be satisfactory.

The present methods and devices allow a very simple procedure with minimal operational steps by the user. In use, the reagent strip 10 is placed in the detector so that the hole 14 in the strip 10 registers with the optics of the detection system. The system 15 / pin 65 previously described, as shown in figures 4 and 5 »works perfectly to obtain the said alignment. A removable cover or other cover is placed over the optics and the strip to protect the assembly from ambient light. This is done to improve the reading of strip 10. Although ** \ the initiation process may start light, sunlight di—

line or high intensity ambient light tends to inhibit the results. Cap 90 ensures that direct light does not reach the reagent strip 10. The cap, which does not need to be impermeable to light, is just sufficient to protect strip 10 from direct light.

The measurement sequence is then started by pressing a button on the measuring device that activates the microcomputer to measure the reflected light from the non-reactivated reagent pad, called an R reading. The cap 90 is then removed and a drop of

S6 CQ blood to the reagent strip 10, usually while the reagent strip 10 is registered with the optics and the reading device. It is preferable that the reagent strip is registered with the optics in order to minimize handling. The cover 90 then closes.

The instrument is capable of perceiving the application of blood or another sample by a decrease in reflectance when the sample passes through the matrix and the reflected light is measured on the opposite side. The decrease in reflectance initiates a timing sequence that is described in detail elsewhere in the present description. Cap 90 must be replaced within 15 seconds of sample application, although this time may vary depending on the type of sample to be measured.

The results are usually presented approximately 3θ seconds after the blood is applied when measuring a blood glucose sample, although a 20 second reaction is acceptable for glucose samples with a glucose concentration below 250 mg / dl. If other samples are to be measured, the appropriate times for presenting the result may differ and can be determined immediately from the selected reagent / sample characteristics.

An exact specific assessment of the level of glucose (or any other analyte to be measured) can be made using the background current, ie the current of the photodetector connected with no reflected light from the reagent pad, to make a second order correction. It has been shown that beyond a period of 2–3 months this value does not change for a specific instrument prepared according to the preferred embodiments of the present description, and it is possible to program this second order reading in the computer's memory as a constant.

However, with a slight change in the procedure, this value can be measured (or normalized) with each analysis for more accurate results. Each LED is activated before placing the blood sample on the reagent strip 10 but with the reagent strip in place. A reflectance value of strip 10 is measured, with the reagent strip 10 in place and with the light shield 90 closed · If this measurement differs from the original measurement of the reflectance value, the power is increased to the LED so that the reflectance is the same · This ensures that the measurement of the blood glucose content is being made with the same repetition scale for each blood glucose reading.

The reason for instituting this method is twofold. First, the intensity of the light-emitting diodes varies widely from LED to LED, even when all LEDs measure— ** · * · *. are new. Second, the efficiency of the LED varies with the temperature * ** rature and life of the LED. With this method, the results are repeatable on the same scale.

The data not worked to calculate a result in a glucose test is a second order current referred to as second order reflectance, R ^, as described above; A reading of the non-reactivated test strip, R, which is about 95% opaque to light and found in S6Cd is also described previously} and a measurement of the end point. Using the preferred embodiments described here, the

ρ 9 end point is not particularly stable and must be accurately timed from the initial blood application. However, the meter as described here performs this time adjustment automatically. For glycose concentrations below 25θ mg / dl, an appropriate, stable endpoint is achieved in 20 seconds and a final reflectance, ^R 20 * ^ is made ^for glucose concentrations above 450 mg / dl A reflectance reading of 30 seconds is appropriate, ^r ^ q · Although the system described here presents a good differentiation up to 800 mg / dl of glucose, the measurement is a little noisy and inaccurate above 450 mg / dl, although not so much that cause significant problems. Longer reaction times provide more appropriate readings for high levels of glucose concentration.

A reflectance reading of 700 nm for dual wavelength measurement is usually done in 15 seconds (R ^). By this time the blood should have completely saturated the reagent pad. After 15 seconds the reaction continues to take place and will be felt, for a small part, by a reading at 700 nm. Therefore, since the absorption of dye by the 700 nm signal is a disadvantage, readings beyond 15 seconds are ignored in the calculations.

The previously reported data not used is used to calculate the parameters proportional to the glucose concentration which can be more easily seen than the reflectance measurements. If desired, a logarithmic transformation of reflectance can be used analogous to the relationship between absorption and concentration of the analyte observed in transmission spectroscopy (Beer's Law). A simplification of the Kubelka-Monk equations, derived especially from reflectance spectroscopy, proved to be particularly easy. In this derivation K / S is related to the analyte concentration with K / S defined by Equation 1

K / St (1) = (1- R * t) ² / (2 XR * t)

R * t is the reflectivity made in a specific end point period, t, e, and the fraction absorbed from the incident light beam described by Equation 2, where R is the reflectance of the final point, R ₂₀ or R ₃₀ .

R * t = (R - R,) / (R - R,) (2) t d dry b

R * t varies between 0 for non-reflected light (R ^) ^and 1 for total reflected light (R) · The use of reflectivity in dry calculations greatly simplifies the meter model as a highly stable source and makes a seen detection circuit unnecessary that these components are controlled with each measurement of R and R,.

dry b

For a simple wavelength reading, K / S can be calculated at 20 seconds (K / S — 20) or 3θ seconds (K / S-30) · The calibration curves that relate these parameters to blood glucose measurements YSI (Yellow Springs Instruments) can be accurately described by the polynomial equation of perceived order represented in Equation 3 ·

YSI = a + a _n (K / S) + a _o (K / S) ² + a _o (K / S) ³ (3) o 1 2 3

The coefficients for these polynomials are listed in Table 1.

TABLE 1

Coefficients for adjusting the third-order polynomial of simple wavelength calibration curves

K / S — 20 K / S — 30 ^to 0 -55.75 -55.25 ^the i 0.1632 0.1334 ^a 2 a _ -5.765 χ io ⁵ Q 2.58 X 10 ” -2.241 x 10 O 1.20 x 10

The simple chemical species to be measured in the preferred embodiments is the MBTH-DMAB inamine dye and the whole matrix to be analyzed is whole blood distributed on a 0.8 µL Posidyne membrane. The magazine entitled “Appli cation of Near Infra Red Spectrophotometry to the Nondestructive Analysis of Foods: A Review of Experimental Results”,

Critical CRC Reviews in Food Science and Nutrition, 18 (3)

203-30 (1983), describes the use of instruments based on the measurement of a difference in Spastic density A.OD λ ^) where 0D \ is the Spastic density of the corresponding wavelength - corresponding to the maximum absorption of a component to be determined and OD) is the density at a wavelength where the same component does not absorb significantly.

The algorithm for measuring dual wavelength is, by necessity, more complex than for measuring simple wavelength but it is much more effective. The first order correction applied by reading at 700 nm is a simple substrate of the blood's own background color. In order to make this correction, the relationship between the absorption at 635 um and 700 bm proper to the blood color can be determined and ended by measuring blood samples with 0 mg / dl of glucose over a wide range of color blood. The color variation was established by variation of the hematocrit, and very linear relationships were observed. From these lines, K / S — 15 at 700 nm was normalized to give equivalence to K / S — 30 at 635 µm. This relationship is referred to in Equation 4, where K / S — 15 is not normalized K / S — 15 at 700 nm.

K / S — 15n = (K / S — 15 x 1.54) - 0.133 (4)

Note that the equivalence of the normalized 700 nm signal and the 635 nm signal is only true for zero glucose. The expressions from which the calibration curves were derived are defined by Equations 5 and 6.

K / S-20/15 = (K / S-20) - (K / S-15n) (5)

K / S-30/15 = (K / S-30) - (K / S-15n) (6)

These curves are better adjusted by equations of fourth order polynomials similar to Equation 3 to which a fourth order term in K / S is added. The computer adjustment «M *» * coefficients for these equations are listed in Table 2.

TABLE 2

Coefficients for adjusting the fourth-order polynomial of dual k / s wavelength calibration curves — 20/15

K / S-30/15

^is the -0.1388 1,099 ^the i 0.1064 0.05235 X 6.259 X io ⁵ —4 1,229 χ 10 - ft -6.12 x 10 -5.83 x 10- ⁸ J -11 _Λ „-11 ^to 4 3.21 x 10 1.30 x 10 It also took place a correction

order to eliminate errors due to chromatography effects. Low hematocrit samples feature low readings at 700 nm as compared to higher hematocrit samples with the same reading at 635 nm. When the ratio of (K / S — 30) / (K / S — 15) is marked as opposed to K / S — 30 over a wide range of hematocrits and glucose concentrations, the resulting line in the graph indicates the boundary between samples that showed chromatography effects (above the curve) and those that did not (below the curve). The K / S— —3θ for the samples above the curve are corrected by rising the reading to correspond to a point on the curve with the same (K / S-3O) / (K / S-15), as shown by the correction made in figure 5 ·

The correction factors mentioned above have been established to adapt a single instrument and a limited number of reagent preparations. The algorithm can be optimized for an individual instrument and a reagent in the same way as described above.

In summary, the system of the present invention minimizes operator actions and provides numerous advantages over prior art reflectance reading methods. When compared to previous methods for determining blood glucose, for example, there are several clear advantages. First, the amount of sample needed to saturate the thin reagent pad is small (usually 5-Ιθ microliter), and is obviously volume independent once the initial blood volume is supplied to the reagent pad. Second, the operator's time required is just enough to apply the sample to the thin hydrophilic layer and close the lid (usually 4-7 seconds). Third, it is not necessary to simultaneously adjust the start time. Fourth, whole blood can be used. The method does not require any separation or use of blood free of red blood cells and likewise can be used with other strongly colored samples. Fifth, by means of normalization and reflectance reading techniques applied in the present invention, the system provides safe, accurate and repeatable readings for the duration of the image exposure system.

Several less obvious advantages arise as a result of the practice of the present invention with whole blood. Normally, aqueous solutions (such as blood) penetrate the hydrophilic membrane to provide a layer of liquid on the opposite side of the membrane, a surface that is not suitable for measuring reflectance. It was concluded, however, that the blood, apparently due to interactions of red blood cells and proteins in the blood with the matrix, wets the polyatomide matrix without an excess of liquid having penetrated the porous matrix to interfere with the reflectance reading in the opposite side of the matrix. In addition, it would be expected that the thin membranes used in the present invention when wet would transmit light and develop only a weak signal for the reflectance measuring device. Previous teachings generally indicated that a reflective layer behind the matrix was needed in order to reflect sufficient light. In other cases, a white pad was placed behind the reagent pad before measuring the color. In the present case, neither a reflective layer nor a white pad is required. In fact, the present invention is normally carried out with a light absorbing surface behind the reagent element when a light incident on the matrix is applied. This is achieved by using a light absorbing surface behind the reagent element, together with the measurement of reflectance at two wavelengths. This allows for acceptable reflectance measurements that are obtained without removing excess liquid from the matrix, thus eliminating a step typically required by previous teachings.

The present invention that is now described in general will be better understood by reference to the specific examples that follow, which are presented for the purpose of just illustrating the present invention and not to be considered limiting the invention unless so specified .

EXAMPLE 1

Reproducibility

A man's blood sample (having a hematocrit level of 45) was used to collect the reproducibility data using the currently preferred embodiment of the system, called the MPX system. The results are shown in tables 3—5 ·

TABLE 3

Reproducibility of a simple wavelength system

Mean (mg / dl) * D. P. (mg / dl)% C.V. «

auotYSI (mg / dl) 20sec. 30sec. 20sec. 30sec. 20sec. 30sec. 25 23.1 23.0 2.1 2.04 9.1 9.0 55 53.3 53.2 3.19 3.32 6.0 6.3 101 101 101 3.0 3.3 3.0 3.0 326 326.6 327 13.3 9.8 4.1 3.0 501 503 17.1 3.4 690 675 28 4.15 810 813 37 4.5 * D.P. «Rf = Standard deviation **% C.V. = Co- variation (measure percentage) YSI Glucose reading in Yellow Spring Instruction—

ment

TABLE 4

Reproducibility of Average a dual wavelength system (mg / dl) 30sec. D.P. (mg / dl) % C.V. YSI (mg / dl) 20sec. 20sec. 30sec. 20sec. 30sec. 25 25 27 1.34 1.55 5.4 5.7 55 55 57.4 2.58 2.62 4.7 4.6 101 101 101.5 2.55 2.18 2.5 2.1 326 332 330 15.0 7.1 4.5 2.1 501 505 21.3 4.2 69O 687 22.8 3.3 8l0 817 30.4 3.7

TABLE $

Reproducibility of a 3.0 mm diameter opening

YSI (mg / dl) mm% c.v.

3.0 mm

55-100

300

600 avg.

4.8 3.0

3.8

3.9

4.9

5.0

5.5

5.1

The blood was divided into aliquots and reinforced—

glucose with limits between 25—800 mg / dl. Twenty determinations were made at each glucose test level from strips taken at random from a sample of 500 strips (Lot FJ4—49B). The results of this study

X ** made the following conclusions!

1. Dual vs. wavelength simple! The average covariation for the 30-second dual result was 3.7% vs. 4.8% for the result of the simple wavelength of 30 seconds, an improvement of 23% for the glucose limits of 25-810 mg / dl. There was an improvement of 33% in the covariation of the glucose limits of 25—326 mg / dl. Here, covariation decreases from 5.4% to 3.6%, a significant improvement in the most used limits. The measurement of the dual wave length of 20 seconds provided improvements in the covariation compared to the measurement of the simple wavelength in the limits of 25—326 mg / dl (Tables 3 and 4)

2. Dual wavelength, result of 20 vs. 30 seconds!

The average covariation for a 20-second result in the limits between 25—100 mg / dl is almost identical to the 30-second reading, 4.2% vs. 4.1%. However, at 326 mg / dl, the 30-second reading has a covariation of 2.1% and the result of 20 seconds is a covariation of 4.5%. As noted in the K / S-20 response curve, the slope begins to decrease just above 250 mg / dl. This leads to poor reproducibility at glucose levels above 300 for the 20-second result. From these reproduction data

- 36 the separation ability for the 20-second result is somewhere between 100 and 326 mg / dl. Later a 250 mg / dl separation was determined from the results of the recovery study developed in Example II.

3 · Size of the opening! Researches were carried out with a smaller opening size of the optic, 3.0 mm. The initial experiment using a disk sample manually moistened in 10 repetitions showed improved covariations with an opening of 3> 0 mm, apparently due to easier registration with the system optics. However, when the machine-made cylindrical membrane was used, the mean (Table 5) of the largest opening, 4.7 mm, was 3 * 9% vs. an average covariation, for the opening of 3 »0 mm, of 5i1% · Es ^ and an increase of 30% was probably due to the irregular surface of the batch of circular membranes as mentioned later.

EXAMPLE II

Recovery!

Blood from 36 donors was tested to compare the present preferred method called MPX with a method characteristic of the prior art using a Yellow Springs Instrument Model 23A glucose analyzer manufactured by Yellow Springs Instrument Co., Yellow Springs, Ohio (YSI) . Donors were divided into men and women and were ordered from 35 to 55% in hematocrit. Blood samples were used within 30 hours of collection, with lithium heparin as anticoagulant. Each sample was divided into aliquots and reinforced with glucose to provide 152 samples in the range of 0—700 mg / dl of glucose. Each sample was tested in duplicate for a total of 304 data points. Response curves were prepared using the appropriate equation (see tables 1 and 2). Then the glucose values of MPX were recorded. The YSI values to provide scanning diagrams, as can be seen in Figures 6a and 6b for the system

single wavelength theme, and 7a through 7d for the dual wavelength system.

Comparison of MPX systems: For the two measurement periods of 20 seconds and 3θ seconds there is visually more dispersion in single wavelength scan diagrams than in dual wavelength scan diagrams

The 20-second reading becomes more dispersed above 250. ** ** mg / dl but the measurement of 3θ seconds does not show wide dispersion until the glucose level is above 50θ mg / dl.

These scanning diagrams were quantified by determining the YSI deviations in various glucose variations. The following results were obtained.

TABLE 6

Accuracy of MPX system from recovery data

Measurement Length of in MPX * D.P. 1 (mg / dl) C. V. 0-50 for variations wave Period (sec) 50-250 250450 Simple 20 15.6 7.2 14.5 Simple 30 / 6.9 7.1 8.8 10.2 Dual 20 12.3 5.3 12.8 - Dual 30 12.19 5.5 8.4

* = Standard deviation

• K4Í = These are covariations inter — methods Note that: The. The system of length dual wave displays results that varied 30% below of the wavelength system simple. B. The system of length single wave, from 0—50

mg / dl, showed a standard deviation of ± 6—7 mg / dl whereas the standard deviation for a dual wavelength measurement was only ± 2.2 mg / dl.

ç. The separation for a 30-second MPX measurement is 250 mg / dl. For the 5θ-25θ mg / dl variation, the two measurements of 20 and 3θ seconds show similar inter-method covariations (approximately 7% for single wavelength, 5.5% for dual wavelength). However, within the limits of 25θ — 45θ mg / dl, inter-method covariations more than double for the 20-second reading to 14.5% for the single wavelength and 12.8% for the dual wavelength

d. The 3-second reading was unusable above 450 mg / dl for both single and dual wavelength measurements (covariations of 10.2% and 8.4%).

In conclusion, the two MPX systems present optimal quantification in the range of 0—45θ mg / dl.

1. MPX system - 3θ seconds dual wavelength: This dual wavelength system provides a 3θ second measurement period with a 95% confidence threshold (defined as the probability of a measurement that is at + 2 Standard deviation of the YSI reading) from a covariation of 11.3% to 50-450 mg / dl (Table 7) and ± 4.4 mg / dl (Standard deviation to 0—50 mg / dl.

2. MPX system - Dual wavelength of 30/20 seconds!

This dual wavelength system provides a measurement period of 20 seconds within the limits of 0—250 mg / dl and a period of 30 seconds for the limits of 250—450. The 95% confidence limits are almost identical to the 3θ seconds dual wavelength MPX system (Table 7), 11.1% covariation for 5θ — 450 mg / dl and +4.6 mg / dl (Standard deviation ) to 0—50 mg / dl.

TABLE 7

Comparison of 95% confidence limits for the MPX system, GlucoScan Plus reagent strips and Accu-Check bG

Variation measurement Simple MPX Wavelength MPX length wave dual mg / dl 20 sec 30 sec · 20 sec, 30 sec 0-50 11.2 mg / dl 13.8 mg / dl 4.6 mg / dl 4.4 mg / dl 50-250 14.4 14.2 10.6 11.0 250-450 17.6 - 11.6 77-405 = GlucoScan Plus (Drexl er Clinic) 15 »9% 77-405 = Accu — Chek bG (Drexler Clinical) 10.7% 50-450 = 20/30 dual hybrid Mon. in MPX System 11.1% 50-450 = Dual wavelength of 30 sec • in MPX system 11 »3%

íííííí

The confidence limits for MPX were found from YSI. The confidence limits for GlucoScan Plus and Accu — Chek bG were found from the healthy vs. negative equation. YSI that eliminates the slope due to small differences in calibration

EXAMPLE III

Stability:

** Most of the level grading work carried out in the optimization of stability has been supplemented with the use of manually moistened 0.8 p Posidyne membrane discs. The specific dye / enzyme formulation was previously established.

1. Ambient Temperature Stability! This study attempted to graphically show any change in the response of the 0.8 p Posidyne membrane reagent stored at 18 ° C - 20 ° C over silica gel desiccant. After 2.5 months there was no noticeable change as measured by the response of a sample to room temperature vs. the response of a sample stored at 5 ° C. Each measurement represented a glucose change of 0—450 mg / dl.

2. Stability at 37 ° £ · A stability study was carried out using the same reagent as the study at room temperature. Differences in the glucose values of the reagent treated at 37 ° C vs. reagent at room temperature, for strips treated with or without adhesive, were graphically represented after the time. Although the data was noisy due to the poor reproducibility of manual strips, the stability was excellent for strips whether treated with or without adhesive.

3. Stability at ^ 6 ° Ci 8 stability studies of 5 to 6 days were developed using different preparations of a similar formulation on a disk membrane (Table 8). For the low glucose test level (80—100 mg / dl) the average glucose value dropped under pressure by 3.4%, the highest drop being 9.55% · For the high test level (280— 320 mg / dl) glucose reading decreased by an average of 3 »4%, the highest decrease being 10.0%.

TABLE 8

PH stability = 4.0, 0.8 µl Posidyne disc reagent

Formulation treated for 5 days at 6 days at 5th ° C % of difference (5 ° C vs. Ambienie Temperature Sample) Sample No Y&I V8Õ-1Ò0 mg / dl) ΪΒΪ (2ÕÔ-320 mg / dl) FJ22B -6.25 + 5.4 FJ27A -4.0 -5 »i4 FJ28B -2.4 -5.3 FJ30H -9.55 -10.0 FJ3IC +4.43 -1.24 FJ36 -3 »2 -8.5

- 4l -

FJ48B * -3.0 0.0

GM48A * -3.0 -2.5

Average of 8

-3.4 ^ These two samples contain twice the normal concentration of enzyme and dye.

A study of the treatment of this membrane at 56 ° C, after a period of 19 days, did not show significant differences for strips treated with or without adhesive. In both cases, the 19-day decrease in glucose value was less than 15% at low test levels (80—100 mg / dl) and also at 3θθ mg / dl.

Another study was completed at 56 ° C using IM a 0.8 µm Posidyne membrane with double the normal concentration of enzyme and dye. Two separate preparations of the same formulation were made and stability was measured after a period of 14 days. The average results of the two studies were compared. Changes between + 0% after the 14-day period were found in both high and low glucose test levels.

EXAMPLE IV

Sample Size

The sample size characteristics for the MPX system are shown in Table 9

TABLE 9

Effect of Sample Size on MPX System Measurements

Simple wavelength

Sample Size (ul)

Dual wavelength

YSI = 56

4l 50 39 31

31 42 30 19 30

TABLE 9 (continued)

4 44 49 49 49 48 4l 45 44 45 44 5 54 48 49 51 50 50 49 48 49 49 10 48 48 50 47 48 54 53 56 55 54 20 49 49 49 50 49 55 57 58 60 58 YSI = 36O 3 301 260 276 286 280 274 232 244 260 252 4 3Ô3 378 367 341 367 361 356 342 318 344 5 398 402 382 370 388 378 387 366 351 370 10 364 362 378 368 368 356 358 379 369 366 20 375 370 38O 378 376 380 382 389 385 384 The referred volumes ! at Table : were transfused

reagent pad 10 depicts in Figure 1, using a micropipette. When blood is applied from a piercing on the finger to a strip, the entire sample cannot be transferred. Thus, the volumes listed here do not represent the size of the entire sample needed to be squeezed from a finger for analysis. A 3 µl sample is the minimum required to completely cover the reagent pad circle. This does not provide enough sample to completely saturate the reagent pad and the MPX system, either at single or dual wavelengths, with poor results. A sample of 4 pl barely saturates the reagent pad, while a sample of 5 µl is clearly appropriate. A 10 µl sample of a large shiny drop and a 20 µl sample is a very large drop and is only likely to be used when the blood from a pipette is used to make samples.

At the lowest glucose concentration, the result of the simple wavelength has some dependence on the sample size, which is completely eliminated using the dual wavelength measurement. Although this pending with the simple wavelength can be considered

43 acceptable, it is clearly undesirable.

EXAMPLE V

ReproducibilityI

The experimental measurements described above were always performed repeatedly, usually 2, 3 or 4 determinations per data point. These sets always showed close agreement even in samples with extreme levels of oxygen and hematocrits. The covariations were well below 5% · It lacks, therefore, that reproducibility is very good to excellent.

The material of the present invention is produced by many commercially or has been described in the literature. The protocols are simple and require little technical expertise and are relatively free from operator error. Tests can be carried out quickly. They use inexpensive and relatively harmless reagents, important considerations for materials used at home. The user obtains results that can be interpreted and used in conjunction with maintenance therapy. In addition, the reagents have a long durability, so the results obtained will be reliable over long periods of time.

The equipment is simple and safe and substantially automatic.

All patents and other publications specifically identified with this description are indicative of the level of expertise of the common experts in the art in which the present invention is included and are here incorporated individually by reference to the same scope, as would happen if each reference were specifically and individually incorporated by reference.

The present invention now being described in

it will be apparent to a person of ordinary skill that many modifications and alterations can be made without departing from the spirit and scope of the present invention as defined in the claims that follow.

Claims (1)

- ia Process for positioning a reagent strip having a notch at one of its ends and a centralized perforation, with reflectance reading equipment that has a rod adjacent to an optical reading device, characterized by! adjusting said notch of the reagent tape against said stem of the reflective reading device, so that said notch can rotate around the stem so that the centralized perforation is already placed on said reading device optics. - 2β Process according to claim 1, characterized in that the reflectance reading device has a channel cascading with said notch, the stem being located inside that channel and the reagent strip also comprising a thin and narrow rectangular blade with a width identical to the said channel and having two ends - 45 ages, said notch being located at the first of said ends, and by additionally placing said rectangular blade in said channel after adjusting the notch to said rod, so that placement in said channel provides adjustment of the reading device optical under the centralized drilling placed in said channel. - 3 ^e Process for aligning a reagent strip having a notch at one of its ends and a centralized perforation with a reflectance reading device having an optical reading device under a channel that is approximately the same width as said reagent tape, that channel also having a rod at one of its ends, characterized by placing the tape in said channel and allowing the adjustment of said notch to the rod, then rotating said tape around the rod so that the centralized perforation is placed on said optical reading device, the reagent tape being well placed in said channel · - 4th Process for timing a measurement on a reflectance reading device, characterized by! at least a first optical reflectance reading is made from a first surface of a porous matrix on a reagent strip, before applying a sample to the second surface of said matrix; at least one additional reflectance reading is made at from said first surface and 46 5? comparing said additional reflectance reading with the first reflectance reading and starting the measurement of time after a predetermined drop in reflectance resulting from the fact that said sample reaches said first surface. 5. ^A method according to claim 4, characterized in that at least one measurement of the reflectance reading is made at a predetermined moment after said additional reflectance reading differs from said reflectance value by the value of the predetermined difference. 6. A method according to claim 5, characterized in that said surface is initially dry and that said additional reflectance reading differs from said first reflectance value when a liquid test medium consisting of an ink mixed with blood penetrates said first surface of said matrix. Process according to claim 6, characterized in that said precursor ink is a combination of glucose oxidase, peroxidase and an MBTH-DMAB dye (3 — methyl — 2 — benzo — thiazolinone hydrazone and 3 — dimethyl — amino— benzoic) · 8. The method of claim 6, wherein said predetermined time is at least 20 seconds. _ 9 to _ Process according to claim 6, characterized in that said predetermined time is at least 30 seconds. Process according to claim 6, characterized in that said predetermined time is a maximum of 30 seconds. - lia Process to normalize the reflective switching in a system that has a Sptico reader capable of reading the reflectance of the light emitted by a diversity of light emitting diodes (LED), this light being reflected to said Sptico reader from a porous membrane, characterized by: exploring that porous membrane in search of a reflectance reading of the intensity of the LEDs before placing a test sample; introducing that reading into the control means, these control means being capable of storing intensity readings. of said LEDs, with the said control means still able to match the difference between a pair of readings of the initial and subsequent LED} and if the said control means are used to activate the power that allows the intensity of the LEDs to be adjusted to provide the same reflectance reading for each LED · - 12th Process for the determination of blood glucose levels characterized by: if a drop of blood is placed on a reagent strip, the said strip is placed on a measuring device by scanning the reflectance in which said measuring device is sensitive to the presence of the blood sample in said reagent strip, starting the measuring device by reflectance exploitation a time sequence after detecting the presence of the said blood sample in the reagent strip} if reflectance measurements are made '· (l) from the luminosity of the said measuring device (R _b ) i (2) before said reagent strip contains blood (R)} and (3) at a predetermined time (R ^)> and calculating if and then R t at which R t = (R —R) / (R -R,)} dry tb using R t to calculate K / S — t, where K / S — t is defined as (lR * t) ² / (2xR * t) ie if glucose levels are calculated from the said K / St value. - 13a - 49 Process according to claim 12, characterized in that the predetermined time is 20 seconds and the glucose level is defined as follows: -55.75 + + 0.1632 x (κ / st) - (5,765 x 10 ^-5 ) x (K / St) ² + (2.58 x 10 ^-8 ) x (K / St) ³ . - 14th _ Process according to claim 12, characterized in that the predetermined time is 30 seconds and the glucose level is defined as follows: —55 * 25 + + 0.1334 x (K / St) - (2,241 x 10 ~ ⁵ ) x (K / St) ² + (1.20 x x10O ^-8 ) x (K / St) ³ . - 15Ô Process for the determination of blood glucose levels characterized by: if a drop of naked blood is placed on the reagent strip) if the said strip is placed on a measuring device by scanning the reflectance, said measuring device being sensitive to the presence of the blood sample on said reagent tape, starting the measuring device by reflectance scanning a time sequence after detecting the presence of said blood sample on the reagent strip) reflectance measurements are made: (l) from the luminosity of the said measuring device (R _b ) j (2) before said reagent strip contains blood ^ sec <? ' ^ ³ ^ 15 seconds after the reagent strip contains blood ^{and at} a predetermined final moment (R ^.) If the readings are used to calculate R * 15 and R * t according to the following properties! ^ ¹⁵ = ^(R 15 - V ^ dry 'VK «t = (R _t - R _b ) / (R _dry - V ί if the K / S and K / S levels are calculated, with ± 5 t being the defined levels of the following way: ^{K / S} 15 ^{= (1_R} * ¹⁵ ) ^{2 / í2xR} * 15) K / S _t = (1-R ^x t) ² / (2xR * t); normalize the said K / S value ^^ to arrive at a K / S value defined by lpn K / S 15n ^{= (1 '54 x K / S} 15 ^} ~ ° » ¹ 333i if a K value S defined by X bl / K / S _{t / JL5} = vs _t - K / S _15n ; and using said K / S ^^^^ value to calculate said glucose levels. - Process according to claim 15, characterized in that the predetermined time is 20 seconds and by defining said glucose level as follows: -0.01388 + O, io64 x ( ^R / ^S t / ₁₅ ) + (6.259 x 10 ^-5 ) xx (K / St / 15) ² - (6.12 x 10 ~ ⁸ ) x (K / St / 15) ³ + + (3.21 χ 10 ^-11 ) x ( ^K / ^S t / ₁₅ ) ⁴ - 17 ^a Process according to claim 15, characterized in that said predetermined time is 3θ seconds and the glucose level (YSI) of the way 51 _4 next; 1.099 + 0.05235 x (K / ^S t / ₁₅ > ^{+ (l} ' ^{229 x 10 x} x (K / St / 15) ² - (5.83 x 10 ^-8 ) x (K / St / 15) ³ + + (1.30 χ ίο ^-11 ) x (K / s _{t / 15} ) \ - Method according to one of the preceding claims, characterized in that said reagent strip consists of a porous matrix} a signal generator system in said porous matrix for reaction with the whole blood sample and a flap connected to said matrix porous to> ** handle said reagent strip after placing the blood sample in the porous matrix. - 19 ^to claim 18 ca be made of dimension with berturas Process according to that characterized by said porous matrix a hydrophilic membrane which contains soes between 0.6 microns and 1.0 microns. - Method according to claim 18, characterized in that said signal generating system consists of glucose oxidase, peroxidase and a coloring indicator - 52 2ia method according to claim 20, ca racterizado in that said dye indicator is a MBTH-DMAB indicator (methyl 3 ^_m ethyl-2-benzo-thiazolinone hydrazone and 3-dimethylamino-benzoic acid). 22. The method of claim 18, characterized in that said porous matrix is maintained in a stable manner with a pH value of less than 4.8 and in an approximately 10% citrate solution. - 23 A method according ^to claim 22, ca racterizado in that said porous matrix is maintained in a stable manner at pH values near 4.0. - Method according to any of the preceding claims for measuring glucose in a whole blood sample in which said reagent strip is adapted for placement in the reflectance reading equipment, characterized in that said strip consists of! a porous hydrophilic membrane having pores with dimensions between 0.6 microns and approximately 1.0 microns, a glucose oxidase, peroxidase signal generator system and a dye indicator incorporated in said membrane for reaction with the whole blood sample) and a flap attached to said porous matrix for 53 handle said reagent strip after placing the blood sample in the porous matrix. - 25 ^to process according to claim 24, ca racterizado for maintaining the reagent ribbon with a pH below 4.8 and in citrate solution to approximately 10%. 26. The method of claim 25, characterized in that the pH value is about 4.0. 27. ^The method of claim 24, wherein said coloring indicator is an MBTH-DMAB indicator. - 28th _ Process according to claim 24, characterized in that the hydrophilic membrane is maintained in a polyamide solution with a positive charge and that it contains at least two smooth sides, the first of said sides being connected to said flap, being possible to place the sample of blood on said first side so that the sample migrates to the second of said sides. Process according to claim 24, characterized in that said flap consists of a rectangular sheet having two ends, the sheet containing a rectangular perforation for central connection of the porous membrane and having said flap. rectangular blade a notch cut at one of said ends. Method according to claim 29, characterized in that said notch is susceptible to adjustment to a stem on the reflectance reading device to ensure repetitive alignment of said membrane on a reflectance reader. Process according to claim 29, characterized in that said hydrophilic membrane contains two smooth sides, the first of said smooth sides of the membrane being connected to said flap in the centralized perforation, and it being possible to insert the blood sample in the first side of the membrane through said perforation so that the sample migrates to the second side of the smooth membrane. 32. The method of claim 31, characterized in that said reagent strip is prepared for reading by the reflectance measuring apparatus after saturation of said second side of the smooth membrane. 33. ^A method according to claim 32, characterized in that said second side of the smooth membrane has an area between approximately 10 nm and 5θ mm 2 34. The method of claim 33, wherein said second side of the smooth membrane has a diameter of between approximately 2 mm and 10 mm. 35. ^A method according to claim 32, characterized in that said second side of the membrane is prepared for reading by the reflectance measuring equipment after placing a volume of approximately 5 microliters and 10 microliters of sample. blood on said hydrophilic membrane. 36a Process according to claim 32, ca 56 characterized in that said second side of the membrane is prepared for reading by said second side of the smooth white without separation of the blood sample in its component parts. - Method according to claim 32, characterized in that said second side of the membrane is prepared for reading after placing a threshold volume of said blood sample on the hydrophilic membrane, the reagent strip being able to read the blood sample despite the volume of that sample on the reagent strip being above that threshold volume. Process according to claim 37, characterized in that said threshold is between 5 microliters and 10 microliters of said whole blood sample. - 39a Process according to one of the preceding claims for the measurement of glucose in a whole blood sample, the reagent strip being adapted to place · * The action in a reflectance reading device, characterized in that said strip consists of! a flap adapted for placement in said reflectance reading equipment, said flap comprising a thin rectangular blade having two ends on each side of a centralized perforation with a diameter between approximately 2 mm and 10 mm, where one of said ends has a notch extending substantially up to the middle from said endj by a porous hydrophilic membrane that has two smooth sides with a first of said sides connected to the flap in the centralized perforation, containing said membrane * pores having an opening with dimensions between 0.6 microns and 1.0 microns and by a dye solution incorporated in the porous membrane reference including glucose oxidase, peroxidase and an MBTH-DMAB indicator, maintaining the said dye solution in the hydrophilic membrane with a pH value close to 4.8 and in a 10% citrate solution, the said reagent strip being capable of measuring taking whole blood samples without separating said sample into its components after having placed a threshold volume of blood sample on said hydrophilic membrane. Process according to claim 39, characterized in that said blood sample can be placed through said centralized perforation on the first side of the hydrophilic membrane and in which the blood sample is liable to migrate to said second side of the hydrophilic membrane, and the readings of the reflectance measurements can be made by the reflectance reading equipment. A method according to claim 40, ca 58 characterized in that said threshold volume is between approximately 5 microliters and 10 microliters of said whole blood sample. The applicant claims the priority of the North American application submitted on April 28, 1988, under serial number 187,602. Lisbon, April 27, 19θ9 · • ÀGEwrí QfieiAL βΑ * POr »> £ BAfií I * 3usr« « 59 SUMMARY PROCESS AND DEVICE FOR THE COLORIMETRIC DETERMINATION OF CHEMICAL AND BIOCHEMICAL COMPOUNDS (ANALYTICAL) IN WATER FLUIDS The present invention relates to a process for determining the presence of an analyte (chemical and biochemical components in aqueous fluids) in a fluid, together with various components of equipment specially designed to carry out the process. «Rf The process involves performing a reflectance reading from a surface of an inert porous matrix impregnated with a reagent that will interact with the analyte (aqueous chemical and biochemical components. **) to provide an absorbent reaction product. light when the fluid to be analyzed is applied to another surface and migrates through the matrix to the surface that The ** will be read. The reflectance measurements are made at two separate wavelengths in order to eliminate interference, triggering a timing circuit by initially decreasing the reflectance by wetting the surface whose reflectance will be measured, due to the fluid passing through the inert matrix. The possibility of repetition is guaranteed through a standardization technique adapted to the light source before each reading, and through an alignment process performed on the reagent strip before the «rf« rf placement in the device. The process and apparatus are particularly suitable for measuring blood glucose levels without having to separate red blood cells from serum and plasma.