MXPA06010066A - Analyte test system for determining the concentration of an analyte in a physiological fluid - Google Patents

Abstract

This invention provides a device for determining the concentration of an analyte like glucose, cholesterol, free fatty acids, triglycerides, proteins, ketones, phenylalanine or enzymes, in a physiological fluid like blood, serum, plasma, saliva, urine, interstitial and/or intracellular fluid, the device having an integrated calibration and quality control system suitable for dry reagent test strips with a very small sample volume of about 0.5µL based on to a new sample distribution system. The production of the inventive analyte test element involves only a small number of uncomplicated production steps enabling an inexpensive production of the strips.

Description

ASSAY SYSTEM TO DETERMINE THE CONCENTRATION OF AN ANALYZE IN A PHYSIOLOGICAL OR AQUEOUS LIQUID FIELD OF THE INVENTION This invention relates to the field of the quantitative analysis of an analyte, for example glucose, in a physiological fluid, for example blood. In particular, this invention provides an analyte assay system and an assay method for the quantitative determination of analytes in a physiological or aqueous fluid, as well as its method of preparation. BACKGROUND OF THE INVENTION The determination of analyte concentrations in physiological samples plays a prominent role in the diagnosis and therapy of various diseases. Analytes of interest include, among others, glucose, cholesterol, free fatty acids, triglycerides, proteins, ketones, phenylalanine, enzymes, antibodies or peptides in blood, plasma, urine or saliva. Normally, fluid from a physiological sample, for example capillary blood, is applied to a test strip to evaluate the concentration of an analyte. Test strips are usually used in conjunction with a measuring device that determines some electrical properties, such as electrical current, if the strip is designed for the detection of an electroactive compound, or to measure the reflectance and / or transmittance of light , if the strip is designed for photometric detection. In systems with optical detection technology, a mixture of enzymes and color generating materials known as cro-ogens is placed on the test strip. The analyte contained in the physiological or aqueous fluid that has been applied to the test strip reacts with the reagents and causes a change in reflectance or transmittance, thus indicating the concentration of the analyte in the test sample. For example, glucose is determined quantitatively by its oxidation to gluconic acid with glucose oxidase. The hydrogen peroxide of the reaction product causes, together with a peroxidase such as horseradish peroxidase, the conversion of a substrate, i.e. of an indicator, into a chromogenic product, which is detectable and related. with the proportion of glucose concentration in the sample fluid. The measurement of glucose concentration in whole blood samples is a very common task. As diabetes causes dangerous physiological complications that lead to loss of vision, renal failure and other serious medical consequences, only strict therapy and disease management minimize the risk of these consequences by adjusting exercise, diet and medication. Some patients often have to check their blood glucose concentration four or more times a day. These patients, as well as doctors and hospitals, need an exact, reliable and ideally inexpensive method to adjust their treatment regimens in order to avoid long-term complications caused by diabetes mellitus. The greater knowledge of diabetes, the acceptance of self-control and self-treatment depend on the availability of adequate devices and have allowed the development of a multitude of devices and methods for personal use and care. Pregnancy, ovulation, blood coagulation, ketone and cholesterol tests are available, for example for a non-exhaustive selection, but the most outstanding thing in the field of self-monitoring is still the detection of capillary blood glucose. In U.S. Patent 4,935,346 a typical device is described for controlling the concentration of an analyte in blood, for example glucose. The method involves taking a reflectance reading of the surface of an inert porous matrix impregnated with a reagent that will interact with the analyte to generate a photoabsorbent reaction product. Most prior art devices are designed to have a measurement zone or chamber where the test sample is introduced directly or via a fluid pathway or channel, the test chamber or membrane contains all the materials necessary for the reactions, which produces a detectable color change of the sample fluid. U.S. Patent 5,430,542 discloses a disposable optical tray and the method for its manufacture. The cuvette comprises two optically transparent waterproof liquid plastic sheets. A third adhesive sheet is positioned between the two transparent plastic sheets and the three sheets are pressed and sealed together. U.S. Patent 5,268,146 discloses a panel of qualitative tests for evaluating a sample for the presence of an analyte containing all the reagents and components necessary to achieve a visible indication of the presence or absence of an analyte in the sample. U.S. Patents 4,761,381 and 5,997,817 disclose devices in which the liquid samples to be analyzed are applied to sample application holes allowing the liquid to enter the capillary channels leading to the reaction chambers, the which contain the material capable of detecting the components of interest in liquids. US Patent Application Publications US 2002 / 0110486A1 and US 2003 / 0031594A1 disclose medical diagnostic devices for fluids that allow the measurement of analyte concentration or characteristics of a biological fluid, in particular the clotting time of the analyte. blood, where such devices have, at one end, a sample orifice to introduce a sample and at the other end a vesicle to extract the sample via a channel to a measurement zone where a physical parameter of the sample is measured and related with the concentration of analytes or with the fluid's characteristics. Due to the various raw materials and large-scale manufacturing processes of these strips there is no guarantee of adequate strip-to-strip reproducibility from one batch to another. Therefore, it is necessary to set a calibration code to each batch of strips that corrects this variability. The calibration code can be marked on the strip container, and the user must enter the code into the meter when a new batch of strips is used. If the user stops entering a new calibration code or enters an incorrect one, the resulting measurement will be incorrect. Some strips of the prior art, for example the strip disclosed in US 6,168,957 B1, are designed to incorporate the calibration code in the strip, thus the meter can read said code before calculating the glucose concentration. The disposable nature of the single-use diagnostic strips allows only destructive testing, due to the consumption of reagents during the determination step, and thus allows only a statistical evaluation of the performance of the batch by the manufacturer, which does not give 100% of certainty of the performance of an individual test strip. And what is even more important, these types of calibration codes carry only retrospective information to the meter or analytical device reader of strips. Thus, a meter can not evaluate the true history of a particular reagent test strip, for example, incorrect storage conditions or a defective packaging, and will generate an error message only if the strip provides totally erroneous and out-of-scale readings in comparison with pre-program data or validation methods. The user can only check and demonstrate the accuracy and functionality of a reagent test strip with specially prepared control solutions at known concentrations provided by the manufacturer. However, this method is also disadvantageous, since the quality check leads to an increase in the consumption of strips and, therefore, to a higher cost. In the same way, this method does not take into account quality variations within a batch. Some of the devices of the prior art integrate positive and / or negative controls that are activated when the sample is added. For example, in the aforementioned U.S. Patent 5,268,146 preferred embodiments of the test device include an integrated positive control and / or an integrated negative control that are composed of other measurement zones containing reagents that cause a visible change in the indicator by themselves or will prevent the change from occurring independently of the presence or absence of the analyte in the test sample. Also, the test device of U.S. Patent 4,578,358 for detecting the presence of occult blood in bodily substances includes positive and negative control zones. An integrated positive or negative control as disclosed in the two previous patents and commonly known from pregnancy tests provides only useful information in conjunction with the panels or strips of qualitative tests and thresholds indicating the presence or absence of an analyte, but it does not make sense for the guarantee of the quality of the quantitative determination of analytes like glucose in the whole blood. In addition, the measurement procedure can be affected by other variable factors in the fluid of the physiological sample. A typical complication in a full blood test is the variability of erythrocyte levels, which leads to results that may not reflect the actual concentration of analytes in the sample. Considering the aforementioned defects, the object of the present invention is to provide a device that has an integrated calibration system, which explains and compensates for any variability that may be generated by fluctuations in the production process or by the variability of the same sample analyzed to assure the user that the test has been carried out properly and that the result is accurate and reliable. Until now, no dry reagent test strip with an integrated calibration system has been disclosed by the prior art, but various prior art publications describe test strips with numerous reaction zones, used to detect multiple analytes or to integrate positive or negative controls as indicated above.

In the US Patent Application Publications US 2002 / 0110486A1 and US 2003/0031594 Al a particularly interesting prior art test strip is disclosed, comprising multiple reaction zones used to guarantee quality but not for a procedure of internal calibration of the strip. The test strip requires a volume of approximately 20 μl of blood and is used to determine the prothrombin time, an important parameter to characterize blood coagulation. However, if a user must perform tests several times a day, as is required for proper management of diabetes mellitus, these large sample volumes are impractical and disadvantageous, especially compared to the state of the art systems. of blood glucose that require only about 1 μl of whole blood but also require, in all cases, a calibration procedure performed by the patient. A reduction in the volume of channels and cavities forming the measuring cavities in the described strip would require complex and expensive production processes, for example "micromolding", which are less suitable for a large scale production of cheap and disposable sensors.

Accordingly, another object of the present invention is to provide an analyte test system for dry reagent test strips that requires not only small volumes of physiological or aqueous fluid but also a production process that does not involve numerous and complicated steps of production and that, therefore, be cheap and usable for products that assist patients in the self-monitoring of blood glucose or other important physiological parameters. SUMMARY OF THE INVENTION This invention provides a device for determining the concentration of an analyte such as glucose, cholesterol, free fatty acids, triglycerides, proteins, ketones, phenylalanine or enzymes, in a physiological fluid such as blood, serum, plasma, saliva, urine , interstitial and / or intracellular fluid, the device having an integrated system of quality control and calibration suitable for dry reagent test strips with a very small sample volume of approximately 0.5 μl based on a new distribution system of samples The production of the analyte test element of the invention involves only a small number of uncomplicated production steps, which makes it cheaper to produce the strips.

Due to the integrated calibration method, the analyte test system of the present invention provides reliable results regardless of blood type, hematocrit level, temperature, etc. In addition, the production variations are compensated by the calibration procedure also integrated. In addition, the aging of the active component is now detectable and can be compensated and / or recorded, which will lead to a shelf life of the product under suitable storage conditions. The present invention provides an analyte assay element for determining the concentration of at least one analyte in a physiological or aqueous fluid of a sample, which has a first and second surface at a predetermined distance opposite each other, both surfaces provided with two substantially equivalent configurations forming high and low surface energy zones that are aligned substantially congruently, to create a sample distribution system with at least two detection zones, in which the applied physiological or aqueous fluid is pushed towards the areas of high surface energy. The sample distribution system contained in the interior of the analyte test element does not have mechanical and / or structural characteristics that resemble walls, grooves or channels for handling and manipulating physiological fluid or other aqueous sample fluids. The analyte assay element is described in various embodiments suitable for a variety of calibration procedures and is adapted to different methods of chemical and analyte determination; it is easily integrated into the test strips used for a single measurement or in more complex arrangements, such as analyte test disks or tapes to provide base units for various measurements. In a preferred embodiment, the analyte assay element provides n predetermined detection zones of said first surface coated with a catalytic formulation that favors the detection of an analyte in a physiological or aqueous fluid, and predetermined detection zones of said second coated surface with n calibration formulations consisting of m white formulations and nm formulations with different levels of the calibration compound, where n is an integer greater than 2, m is an integer equal to or greater than, and n > m, configured proximal to the center of the analyte assay element, which enables the detection means to obtain the results of the predetermined 2n detection zones and which subsequently allows the processing means to calculate the nm calibration coefficients of a polynomial equation of calibration that obeys a regression coefficient to validate the quality of the n-m calibration coefficients calculated from the calibration equation, and the determination of the unknown concentration of an analyte in a sample of physiological or aqueous fluid. In another aspect, the invention provides a method for preparing the analyte assay element of the present invention with the steps of: generating high and low surface energy zones on a base layer having a first surface, forming the high areas surface energy a hydrophilic access path with n predetermined detection zones, where n is an integer greater than 2, generating a corresponding configuration of high and low surface energy zones on a coating layer having a second surface, applying a formulation catalyst on the n detection zones of the first surface, said catalytic formulation promoting the detection of a concentration of analytes contained in a sample of physiological or aqueous fluid by means of transmission photometry or absorbance, applying n calibration formulations on the n detection zones of the second surface, constituted said n formulations s calibrated by m white formulations and n-m formulations with different levels of the calibration compound, m being an integer of at least 1, and n > m, which is identical or substantially equivalent to the analytes and is capable of causing the same chemical reaction in the catalytic formulation as the analyte in the physiological or aqueous fluid sample, laminating the layers of the first and second surfaces in opposite zones of a central layer having a gap that provides a cavity for the sample distribution system formed by the high surface energy zones on the first and second surfaces of the base and coating layers, drilling or cutting the laminated sheets in their final form . Other features and advantages of the present invention and of the preferred embodiment thereof will become apparent from the following description together with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a perspective view of an embodiment of the analyte test element of the present invention in the form of a test strip. Figure 2 is a perspective view of the embodiment according to Figure 1, showing the expanded sample distribution system. Figure 3 is an exploded perspective view of the device according to Figure 1 showing the three layers separately. Figures 4a, 4b and 4c show different forms of the discontinuity of the core layer forming the sample cavity together with the first and second surfaces. Figure 5 is a section of a detection zone of the sample distribution system constructed by hydrophobic guide elements. Figure 6 is a section of another embodiment of a detection zone of the sample distribution system using hydrophilic access routes. Figure 7 shows different embodiments of the distribution system of samples with different configurations of access roads and detection zones suitable for different calibration methods.

Figure 8 shows the sample distribution system of Figure 6 together with a detector and light emitting means in a section. Figure 9 is a graph showing the calculation of analyte concentration in the sample using the standard addition method. Figure 10 is a graph showing the validation method for the calculated result and the calibration data. Figure 11 shows different forms of the analyte test strip. Figure 12 shows a typical application of a test strip of the invention with a meter. Figure 13 shows the analyte test system with an inserted analyte test strip. Figures 14a, 14b and 14 c show the construction of an analyte test disk. Figure 15 shows an analyte test disk comparing it with an analyte test strip. Figures 16a and 16b show an analyte test system with an integrated analyte test disk. Figures 17a and 17b show an analyte test system with an analyte test strip on the left side and the right side management mode.

Figure 18 shows an analyte test tape and a folding tape to build a package. Figures 19a, 19b, 19c and 19d show the steps of producing the analyte test elements in strip form. The layers shown in Fig. 5, and 8 are not to scale, in particular the thickness of the layers 16, 17, 18, 19 is greatly exaggerated. DETAILED DESCRIPTION OF THE INVENTION As shown in Figs. 1 and 2, the analyte test strip 1 of the present invention is a multilayer array comprising a base layer 2, a core layer 3 covering the base layer 2 and a covering layer 4 covering the central layer 3. The central layer 3 has a discontinuity 5 which creates a hollow cavity together with the base layer 2 and the covering layer 4. Within said cavity there is a distribution system for Samples 6 connected to a sample application zone 9 located on one side of the analyte test strip. The area of application of samples 9, as a contact surface with the user, is preferably formed by a convex curve 10 extending from a main side of the analyte test strip for easier application of the sample. Opposed to the sample application area 9, 10 on the second main side of the analyte test strip is an air vent 11 which allows air displacement while distributing the physiological or aqueous fluid to the predetermined detection zones. 6a, 6'a (see Fig. 3). It can be seen that the construction requires only air ventilation, independent of the number of predetermined detection zones used within the analyte test element. The described elements of the system of distribution of samples with zones of high superficial energy, zone of application of samples, ventilation of air, central layer and discontinuity in the central layer form the totality of the analyte test element, which creates the capillary action intrinsic to carry out the distribution of the physiological or aqueous fluid applied to the predetermined detection zones. In addition, the analyte test strip 1 has registration characteristics 7, 8 useful for differentiating between various types of analyte test strips in the determination of different analytes. By these means, a multi-analyte meter could be instructed to start a special program or procedures with selectable parameters upon insertion of the strip necessary for the determination of a particular analyte. As illustrated in Fig. 3, which represents the multilayer arrangement of Fig. 1 and Fig. 2 in an exploded view, the base layer 2 provides a first surface 2a, and the covering layer 4 provides a second surface 4a. The first surface 2a and the second surface 4a are structured with zones that will create the sample distribution system 6. The configuration of the sample distribution system 6 comprises a predetermined number of analyte detection zones 6a and sample access routes 6b , which are aligned and registered mainly in a manner consistent with the assembly of the multilayer layout. The central layer 3 determines the distance between the first surface 2a of the base layer 2 and the second surface 4a of the covering layer 4 and has a discontinuity 5 to form a hollow cavity together with the first surface 2a of the base layer 2 and the second surface 4a of the coating layer 4. The sample distribution system 6 which will be formed between the first surface 2a and the second surface 4a is placed inside the cavity created by the discontinuity 5 of the central layer 3 and the first surface 2a of base layer 2 and second surface 4a of coating layer 4. Preferably, the hollow cavity is substantially larger in design than the sample distribution system.

With the purpose of the discontinuity 5 of the central layer is only to create a cavity for the sample distribution system 6, the discontinuity 5 of the central layer 3 can have different shapes, examples of which are shown in Fig. 4. Fig. 4a shows a cavity of the umbrella-shaped analyte test element 12, Fig. 4b shows a cavity of the rectangular analyte assay element 13 and in Fig. 4c, the sample cavity 14 has a circular shape. The discontinuity 5 of the central layer 3 does not affect the size of the predetermined detection zones 6a or the size of the access routes 6b of the sample distribution system 6 and, therefore, does not influence or change the volume of shows necessary. In comparison with the sample distribution system 6, the shapes of the cavities shown in Fig. 4 are rather simple, thus allowing the application of simple tools for drilling and rapid processing with lesser demands in reference to the accuracy of the record.

The sample distribution system 6 located in the cavity formed by the discontinuity 5 of the central layer 3 and the first surface 2a of the base layer 2 and the second surface 4a of the covering layer 4 is composed of zones of formation of high and low surface energy on said surfaces 2a and 4a. The areas of high and low surface energy on the first surface 2a of the base layer 2 and the second surface 4a of the covering layer 4 are aligned and basically arranged in a congruent manner with respect to each other. As the applied physiological fluid, or any other aqueous sample, moistens only the high surface energy zones, it is then forced into the predetermined flow paths 6b and to the detection zones 6a of the sample distribution system 6 and between the first surface 2a of the base layer 2 and the second surface 4a of the cover layer 4. Fig. 5 shows a construction of the sample distribution system 6 using hydrophobic "guide elements". In this embodiment of the analyte test element of the present invention, the base layer 2 and the cover layer 4 are coated by a hydrophobic layer 16, except for the zones that will form the access routes of the samples and the detection zones. The hydrophobic layer 16 creates a zone of low surface energy, which will exert a repellent force on an applied sample fluid and, therefore, will force the sample fluid into the high surface energy zones that will form the sample distribution system.

Preferably, the hydrophobic layer 16 is applied on a hydrophilic surface, which is wettable by the physiological or aqueous fluid and transparent to light, in particular to UV, carcano IR and / or to the visible range of the electromagnetic spectrum. The process described above requires a hydrophilic surface, which can be produced from a natural hydrophilic polymer such as cellophane or glass, as well as from hydrophobic surfaces of common polymers (examples are given below) by surface conversion hydrophobic in hydrophilic thanks to a coating process or to the physical or chemical deposition of plasma in hydrophilic monomers that can be vaporized under vacuum, for example, ethylene oxide, ethylene glycol, pyrrole or acrylic acid. Subsequently, the configuration of the "guide elements" can be performed by printing hydrophobic ink on the hydrophilic surfaces of the base and coating layers. A suitable hydrophobic ink will have contact angles with water normally of more than 70a and a surface energy normally of less than 33 mN / m and contains monomers, oligomers and prepolymers with hydrophobic functions, such as isooctyl acrylates, dodecyl acrylates, derivatives of styrene or systems with partially fluorinated carbon chains. Fig. 6 shows another construction of the sample distribution system through the use of hydrophilic access routes. In this embodiment of the analyte test element, the base layer 2 and the cover layer 4 are coated with a hydrophilic layer 17 which creates, by this means, areas of high surface energy. The hydrophilic agent printed on the hydrophobic surface is very wettable by a physiological or aqueous fluid; thus, the high surface energy zones that create the hydrophilic access routes of the sample distribution system will exert a positive capillary force on the physiological or aqueous sample fluid applied to transport the sample fluid to the separate detection zones. The hydrophilic configuration can be carried out by printing a crosslinkable and / or partially insoluble hydrophilic or amphiphilic agent on a hydrophobic surface. Inks with hydrophilic functions can be obtained from a wide selection of crosslinkable water-soluble polymers; Acrylate derivatives prepared from polyalcohols, polyethylene glycols, polyethylene oxides, vinylpyrrolidone, alkylphosphocholine derivatives and others are particularly useful; organically modified silicone acrylates, which are a crosslinkable species of modified organopolysiloxanes are also particularly useful. Suitable coatings provide a typical water contact angle of less than 252 and a typical surface energy of more than 70 mN / m. The base layer 2 and the coating layer 4 suitable as a substrate for the printing process can be composed of a material such as glass, polyvinyl acetate, polymethyl methacrylate, polydimethylsiloxane, polyesters and polyester resins containing fluorene rings, polycarbonates and polycarbonates. polycarbonate-polystyrene graft copolymers, terminal modified polycarbonates, polyolefins, cycloolefins and cycloolefin copolymers and / or olefin-maleimide copolymers. In case the substrate has an intermediate hydrophobic character, the printing of the hydrophilic access routes with a surrounding hydrophobic configuration, ie, a combination of the constructions of Figures 5a and 5b is also possible. In an alternative embodiment, the first or second surface is provided with the hydrophilic / hydrophobic configuration (6, 14) while the corresponding surface provides a homogeneous configuration of hydrophilic pixels surrounded by a hydrophobic zone which thus creates a surface of semihydrophilic character and semihydrophobic (amphiphilic character), which eliminates the need to align the hydrophilic and hydrophobic configuration (6, 14) of the first surface with a hydrophilic and hydrophobic equivalent configuration (6 ', 14') of the second surface. The properties of this amphiphilic surface can be easily designed according to the geometric configuration of the hydrophilic pixels and the overall ratio between the hydrophilic zone and the hydrophobic zone. In the disclosed invention, the amphiphilic character, respectively the ratio between the hydrophilic pixels and the hydrophobic zones, is designed so that the sample fluid progresses from the hydrophilic pixel to the hydrophilic pixel only if the opposite surface provides the hydrophilic character. If the opposite surface provides the hydrophobic character, the movement of the fluid within the capillary range of the analyte assay element will be interrupted. This mechanism allows the above-described method to form a functional analyte test element without the strict requirement of an accurate record of the corresponding configuration of the sample distribution system provided on the first and second surfaces.

Preferably, however, both the first and the second surfaces are provided with equivalent configurations of high and low surface energy to ensure rapid distribution of the sample fluid within the hydrophilic access routes of the sample distribution system. In addition, it is possible to physically raise the high surface energy zones of the first and second surfaces of the low surface energy areas by etching, embossing or simply printing the hydrophilic layer (17) with an increased thickness in about three to five times with respect to the first and second surfaces. Due to this elevation, the capillary interval of the hydrophilic access ways is reduced with respect to the surrounding area and exerts a greater capillary force on the sample liquid. The volume required for the sample distribution system contained in the analyte assay element of the preferred embodiment is very low, approximately 0.5 μl-1.0 μl and requires only about 100 ni-150 or per detection zone if the zones of high and low surface energy are created by hydrophobic guide elements or hydrophilic access routes or by a combination of both. However, it will be obvious to a person skilled in the art that the volume of the sample distribution system will vary with the various designs and with the number of predetermined detection zones employed. As stated above, the creation of a sample distribution system with a volume like this, which includes multiple access routes for the distribution of samples and detection zones, is very difficult or almost impossible with strip technology. of prior art prior art. The amount of physiological sample necessary to perform a measurement on the analyte test element of the present invention is as small as, for example, 1/40 of the amount that is required for the operation of the device described by Shartel et al. . in the publications of the US Patent Applications US 2002 / 0110486A1 and US 2003 / 0031594A1 and, for example, 1/10 of the volume of the microcuvettes of the prior art (HemoCue Glucose Systems). Fig. 7 shows different configurations of the sample distribution system that can be made by hydrophilic access routes, as illustrated in Fig. 6, or by hydrophobic "guide elements", as illustrated in Fig. 5, or by a combination of hydrophilic access routes and hydrophobic guide elements. Box Al in Fig. 7 illustrates all the cases for the simplest system of sample distribution. Column A of Fig. 7 shows the formal design of the sample distribution systems without background correction, while column B provides the designs for the distribution systems of samples with background corrections, column C indicates the order highest of the polynomial calibration equation realizable with adjacent designs, and column n indicates the necessary number of predetermined detection zones of each surface according to the number of measurements needed. The literals in each design indicate the position of the background correction (c), sample (1), and all the associated calibration zones (2, 3, 4, 5, 6) with the increasing amount of the calibration compound. The simplest calibration is represented by a linear equation in which the relationship between the measurement and the concentration of analytes is strictly proportional. The calibration of the analyte test element is generally carried out using the standard addition method by adding the calibration compound of the different calibration zones to the sample and the subsequent calculation of a linear or monotone nonlinear calibration equation. Fig. 9 gives a more detailed explanation about case I. The calibration or order model (column C) needs to be appropriate for the selected analyte and for the detection chemistry used., therefore it is not possible to apply a linear calibration model to a chemical reaction that obeys a fourth order model and vice versa. However, it is still possible to use the analyte assay element designed for five standard additions as a linear calibration, the higher number of standards will allow even more accurate measurement and statistical validation with greater significance in terms of correlation coefficient, standard deviation and standard error of the test compared to a linear calibration based on two standards. In addition, a repetition of the sample and standard measurements is also possible, thus two independent linear calibrations can be performed per particular sample of physiological or aqueous fluid with the embodiments shown in row IV. In the same way, it is possible to use the same analyte assay element for the determination of two analytes. Conversely, a multi-channel system can be performed within the same set of predetermined detection zones if the selected detection chemicals do not cause interference problems, so the educts and reaction products of one reaction will not participate in the other reaction and the produced indicator dye absorbs light in a range of substantially different wavelength. With respect to FIG. 3, the analyte detection zones 6'a of the sample distribution system 6 of the first surface 2a of the base layer 2 are characterized in that they are coated with a catalytic formulation 18, as shown in FIG. Figures 5 and 6. The catalytic formulation 18 contains, as reactive components, a promoter that undergoes a catalytic or non-catalytic reaction with the analyte, if necessary, together with a coenzyme, and an indicator that generates an optically detectable product, which thus allows to detect the analyte contained in the sample 15 by transmission photometry or absorbance. Preferably, the promoter is an enzyme selected from the group consisting of dehydrogenases, kinases, oxidases, phosphatases, reductases and / or transferases. The optional coenzyme contained in the catalytic formulation is a necessary substance for certain enzymes that facilitates the enzymatic reaction, the adenine-3-nicotinamide dinucleotide is necessary, for example, for glucose dehydrogenase. In a test system to determine the glucose concentration, the glucose in the sample is oxidized by oxygen and glucose oxidase to form gluconic acid and H202. Then the amount of H202 produced quantitatively is measured by Reaction (1) or the Reaction (2). Reaction (1): POD Dye (colorless) + H202 Oxidized dye (colored) + H20 Reaction (2): H2O2 + Fe2 +? Fe3 + + H2O Fe3 + + dye - complex dye-Fe3 + In Reaction (1), the enzyme peroxidase (for example, horseradish peroxidase, icroperoxy asa) catalyzes the oxidation of the dye and converts H202 into H20. The intensity of the color is directly proportional to the concentration of glucose in the sample. Representative examples of dyes include o-dianisidine, 4-aminoantipyrine and 3, 3 ', 5, 5'-tetramethylbenzidine. In Reaction (2), H202 oxidizes Fe2 + to Fe3 +. Fe3 + forms a chelate colored complex with a specific absorption peak. Representative examples of ferrous salt include ferrous sulfate and potassium ferrocyanide. Representative examples of chelate dye include xyleneol orange. The amount of Fe3 + chelate complex formed is proportional to the amount of glucose in the sample.

Another test used to determine glucose concentrations in physiological fluids is shown in Reaction (3); here the glucose dehydrogenase (GDH) reacts specifically with the glucose of the sample in the presence of a coenzyme (dinucleotide adenine-3-nicotinamide (3-NAD)) to form NADH, the reduced form of 3-NAD. The NADH subsequently reacts with an electron-accepting dye, for example 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT), catalyzed by the enzyme diaphorase, to generate a purple- dark reddish The intensity of the color measured at 640 n is directly proportional to the concentration of glucose in the sample. Reaction (3): GDH Glucose + 3-NAD * Gluconic acid + NADH NADH + MTT diaphorase (yellow) > MTT (purple-reddish) + 3-NAD An alternative possibility to wild-type GDH is pyrroloquinoline-equinone-glucose-dehydrogenase (GDH-PQQ) which is often used for the electrochemical determination of blood glucose, but which could be used in an optical detection method by use of indicator dyes formed by a reduction similar to MTT or by indicators of chelate complexes as shown in the last half of the reaction scheme (2). However, the catalytic formulation 18 may contain glucose oxidase or gluscosa dehydrogenase if the device is intended for the determination of the concentration of glucose in a physiological fluid. Accordingly, the enzyme contained in the catalytic formulation can be cholesterol oxidase, when the analyte is cholesterol; acohol-oxidase, when the analyte is an alcohol; lactate oxidase, when the analyte is lactate, and the like. Suitable indicators for generating an optically detectable product, either alone or in combination with other chemical compounds, and together with a suitable promoter, for example, an enzyme, are preferably selected from the group consisting of aromatic amines, aromatic alcohols, azines, benzidines, hydrazones, aminoantipirines, conjugated amines, conjugated alcohols and aromatic and aliphatic aldehydes. Specific examples of indicators include 3-methyl-2-benzothiazolinone hydrazone hydrochloride; 3-methyl-2- (sulfonyl) -benzothiazolone-hydrazone hydrochloride (MBTH); 8-amino-1-naphthol-5,7-disulfonic acid (Chicago acid); 3,3 ', 5, 5'-tetramethylbenzidine (TMB); 4, 5-dihydroxy-2,7-naphthalenedisulfonic acid; l-hydroxy-2-naphthalenesulfonic acid; N, N-dimethylaniline; o-tolidine; 3-dimethylaminobenzoic acid (DMAB); ionic acid salt 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS); 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT); and / or 3,5-dichloro-2-hydroxybenzenesulfonic acid In case the chemical compound is an acid, a water-soluble salt of such an acid, for example an ammonium salt, can be used in the preparation of the formulation Catalytic A variety of suitable enzyme dyes are available as a catalytic formulation from prior art publications, for example Wong et al (U.S. Patent 6,312,888); Philips et al. (U.S. Patent 4,935,346) and Berti et al. (U.S. Patent 4,247,297). Catalytic formulations suitable for the present invention are based on a non-reactive base, the indicator components (dyes) and an enzyme or combination of enzymes as a promoter. The non-reactive base provides a vehicle, which needs to be suitable for the coating process, preferably to ink jet printing, stabilization of enzymes and fixation of the enzyme and the indicator system on the surface of the detection zones. A typical composition for 100 ml of formulation is provided below.

Non-reactive base: Distilled water 65 ml Citric acid 2.4 g Buffer system Sodium citrate -2H20 3.2 g Buffer system Polyethylene glycol 1.0 g Coating inhibitor N-Methylpyrrolidone 2.0 g Co-solvent for some indicator dyes (optional) BAS 3,0 g Stabilization of enzyme Gafquat 440 (ISP) 1,0 ml Advantage S film-forming agent (ISP) 1,0 g PVA film-forming agent (low molecular weight 1,5 g Stabilization of enzyme Adjust the pH to 6.5 and fill up to 100 ml Catalytic formulation: (all components are added to 100 ml of the non-reactive base) GOD (Aspergillus niger) 2.0 g (250 U / ml) POD (horseradish) 2.0 g (250 U / ml) Indicator system a) TMB 0.801 g Indicator system b) ABTS 0.915 g Indicator system c) MBTH 0.719 g DMBA 0.551 g Indicator system d) MBTH 0.359 g Chicago acid 1.064 g The catalytic formulation may be composed of indicator systems a) to d) in combination with various hydrogen peroxide producing enzymes such as GOD. Although the pH of the non-reactive base formulation needs to be adjusted to the requirements of a new enzyme if GOD is replaced by another catalyst. Examples of reactions not catalyzed by an enzyme are the detection of albumin with tetrabromophenol blue and the detection of ketones with a mixture buffered with glycine phosphate and nitroprusside in the visible range of the electromagnetic spectrum. If the analyte assay element is designed to perform n determinations, where n is an integer greater than 2, all n detection zones 6'a in the first surface are coated with the catalytic formulation 18 that promotes detection of the analyte. in the physiological sample.

With respect again to FIGS. 3, 5 and 6, the detection zones 6a of the second surface 4a of the coating layer 4 are characterized in that they are coated with a calibration formulation 19 comprising a calibration compound. Preferably, the calibration compound contained in the calibration formulation 19 which overlies the predetermined detection zones 6a of the second surface 4a is identical or substantially equivalent to the analyte and is capable of inducing the same chemical reaction in the catalytic formulation as the analyte in the sample of physiological fluid. In case the analyte of interest in the physiological sample is glucose, then the calibration compound is preferably also glucose. The non-reactive base as described for the catalyst formulation is suitable for the calibration formulation as well and requires only the addition of the necessary levels of the calibration compound. N-methylpyrrolidone, the necessary cosolvent for some of the indicator dyes, can be omitted. The exact dosage of the calibration compound applied to the different detection zones is critical for a suitable calibration procedure and, therefore, for a reliable calculation of the concentration of the analyte in the sample fluid. Therefore, just like the catalytic formulation, the calibration formulation also preferentially covers the predetermined detection zones by means of ink jet printing. By this means it is possible to accurately dose the quantity of calibration compound and apply it over a specific detection zone. If the analyte assay element is designed to perform n determinations, where n is the number of determinations without repetitions of the background measurements, which is an integer greater than 2, then n predetermined detection zones on the second surface 4a are coated with n calibration formulations composed of nm formulations with different levels of the calibration or analyte compound and m blank formulations, where m is an integer of at least 1 and n > m. In other words, at least one of the n detection zones of the sample distribution system does not contain the calibration compound to allow the determination of the analyte concentration. After having applied the physiological fluid to the area of application of the sample and have distributed it in the predetermined detection zones by capillary action, the catalytic formulations are dissolved in the n predetermined detection zones of the first surface 2a as well as the n calibration formulations over the predetermined n detection zones of the second surface 4a forming a mixture of analytes, the calibration compound (which can be another analyte), the promoter and the indicator dye. Within these n mixtures, the optical density changes proportionally to the different levels of the calibration compound plus the unknown level of analyte, thus allowing the optical determination of the n results by transmission photometry and / or absorbance and the calculation of the concentration of analyte Preferably, the catalytic formulation and the calibration formulations applied to the predetermined detection zones are readily soluble in a physiological fluid and / or water and are positioned. one near the other to allow a fast diffusion mixture of both components, allowing them a rapid reaction of all the components contained in the detection zones to accelerate the rapid photometric determination of the analyte concentration. Fig. 8 shows a detector arrangement for measuring the optical density of the sample within the analyte test element according to Fig. 6. The arrangement includes a light source 20, which emits light 24 of a certain wavelength in the direction of the detection area of the sample. The light emitted from the light source 20 passes through an optical device 21, for example a diffuser or lens, and through an opening 22, through the base layer 2, through the sample 15 and through the coating layer 4 of the. detection zone and is detected on the opposite side of the device thanks to a detector means 23. Because there are more than two detection zones disposed within the sample distribution system, thanks to which two of the detection zones contain levels However, although the known, but different, components of the calibration compound, it is possible for the processing means to calculate the unknown concentration of the analyte from the n measurements made with the physiological fluid in the analyte test element. Fig. 9 shows a typical calculation of an analyte concentration in a sample by the standard linear addition method, known calibration technique used in various fields of analytical chemistry, but now integrated and used with a dry reagent test strip for the first time. In this example, the sample distribution system includes three analyte detection zones, two are coated by different predetermined levels of a calibration compound. After applying the physiological fluid to the sample distribution system, the catalytic reaction occurs in the analyte detection zones and the light emitter and meter detection arrangement measures a first optical absorbance 25a of the sample located in the area of detection with the first level of the calibration compound. The reading of this detection zone represents a signal proportional to the combined concentration of the first calibration compound and the concentration of the analyte. In parallel, a second optical absorbance 26a of the sample located in the detection zone is measured with the second level of the calibration compound representing a signal proportional to the combined concentration of the second calibration compound and the concentration of the analyte. In addition, a third optical absorbance 27a is determined from the detection zone containing only the sample with the unknown concentration of the analyte. Since there is a linear correlation between optical density and analyte concentration, following the Lambert-Beer Law, the analyte test system's processing medium can calculate, by linear regression analysis of the measurements, the coefficients for the equation calibrated y = c0 + cix in the previous examples. The concentration of analyte in the sample of physiological or aqueous fluid is determined by the zero point (y = 0) 28 of the previously calculated calibration equation. A general representation of the calibration equations to be applied in the form of: H-1 is provided,? n-?) and C («- 1) X with y = f (results of the optical measurement); x = f (concentration of the calibration compounds); n number of measurements necessary for the determination without repetitions or background measurements according to Fig. 7. This polynomial equation format provides, together with the n-values presented in Fig. 7, the set of calibration settings plus useful for the different designs of the sample distribution systems in the aforementioned figure. The values for x and y can represent data calculated by a function to allow preprocessing of the raw data generated by the detection means. Thus, it is possible to use a logarithmic function for the linearization of the raw data. It would have to be obvious from the disclosure that the invention is not limited to the designs of the sample distribution systems of Fig. 7; and a person skilled in the art is capable of designing a system with n greater than 6 together with the information supplied. A detailed introduction in the standard linear and nonlinear addition methodology is provided by Frank et al. (Anal. Chem., Vol. 50, No. 9, August 1978) and Saxberg et al. (Anal. Chem. Vol. 51, No. 7, June 1979).

A preferred embodiment of the analyte assay element of the present invention according to Fig. 3 is intended to comprise a detection zone that includes the catalytic compounds but no calibration compound (6ax and 6 'ai, res.), An area of detection including the catalytic compounds and a first concentration of the calibration compound (6a and 6'a2, res.), a detection zone including the catalytic compounds and a second concentration of the calibration compound (6a3 and 6'a3, respectively) .) and a detection zone for bottom absorption (6c and 6'c, res.). Thanks to the last detection zone, which does not include any calibration compounds or catalytic compounds, it is possible to determine the background absorption of the sample, for example hemoglobin in the case of whole blood, and to take it into account during the calibration process. Fig. 10 illustrates a preprogrammed validation method for the calculated results and the calibration data by which the validity of the measured results is verified by defining a "validation window" 29 for valid and correct measurements. By this means, the analyte test system can force all data towards a validated and useful concentration range, for example 30 to 600 mg / dl for glucose, and a valid range for optical density, for example 0, 1 to 0.9. Similarly, the processing means can force the slope and intercept or, more generally, the coefficients c0 to c (n-i) to a valid range, which is particularly useful for non-linear polynomial equations. Fig. 10 shows a population of valid measurements with a corresponding calibration line located within the limits of the validation window 29; see literals 25b to 27b and 30. Validation of results is even more powerful through statistical evaluation and linear regression analysis. The quality of the calibration can be judged by a correlation coefficient r2 and a confidence interval, so the analyte test system can refuse to show a measurement result if the correlation coefficient falls below a preprogrammed threshold. Alternatively, the processing means can calculate a range of tolerance or concentration of the result based on the calculated confidence interval. These methods allow a high quality control of the results provided to the patient, which is used and is known today only from sophisticated and expensive laboratory methods and equipment. It is even more important for the patient / user, especially in hospital environments, the right to have quality assured at the time of measurement.

Greater security is possible in another embodiment of the present invention; here the analyte test system is configured to relate the concentration of an inert dye to the amount of the calibration compound used in the calibration step. The calibration formulation is composed of the calibration compound and the inert dye at a pre-established ratio and fixed with respect to each other before being dosed in the predetermined detection zones of the analyte test element. Thus, the processing means of the analyte test system has the ability to trace and correct small variations in the deposited amount of calibration compound when the detector means is configured to determine the concentration of the inert dye with a wavelength different from the wavelength used to evaluate the reaction of the indicator compound with the analyte. Furthermore, the control of the manufacturing process of the dosage and coating step of the calibration formulation becomes observable and, therefore, more reliable. Said inert dye is preferably a water soluble dye selected from the group consisting of bright black BN; bright blue G; Carmoisine; coumarin 120; direct blue 2B; indigo carmine; cochineal red; ponceau 4R; rhodamine 19; sunset yellow; tartrazine; and / or a water soluble derivative of malachite green. Due to the integrated calibration method and the validation method, the analyte test system of the present invention provides reliable results by compensating for endogenous interferences, such as different blood types and hematocrit levels, as well as exogenous interferences, such as nutritional supplements such as vitamin C or medications, which, otherwise, would influence and modify the results of the measures. As the calibration of the analyte test system is performed in parallel to the measurements, different environmental parameters such as the temperature existing at the time of measurement have no consequences on the accuracy of the determined results. In addition, the production variations, for example the variations in the thickness of the central layer, are compensated by the integrated calibration procedure and for the aging of the active component, for example the loss of its enzymatic activity, it is ponderable and it can be compensated, which leads then to a life - prolonged useful in deposit of the product. Fig. 11 illustrates different embodiments and forms of analyte test strips of the present invention adapted to different analyte test systems.

Fig. 12 shows the insertion of the analyte test strip in an analyte test system. In a preferred embodiment, the analyte test strip is designed to have a lateral and concave extension 10 located on the larger side of the test strip, where the application zone of sample 9 resides. This feature allows for easy application of capillary blood samples from the arm or finger of patients, as shown in Fig. 13. In another embodiment of the present invention, as shown in Fig. 14, multiple analyte test elements they are symmetrically arranged around a central point to form an analyte test disk 31 with outwardly facing sample application zones 39. The typical analyte test disk 31 according to Fig. 14a includes nine analyte test elements of the present invention. As shown in the exploded view of Fig. 14b, the analyte test disc 31 is coated with a disc cover composed of an upper layer 32 and a lower layer 33. The lower layer of disc cover 33 can be also provided with a moisture absorbing layer 34. The upper layer 32 and the lower layer 33 of the disk cover have interruptions arranged in a congruent manner with respect to each other forming an optical window 35 in which the test element is located. of analytics used for the current measurement procedure. Adjacent to the optical window 35 are provided in the outer peripheral areas of the upper layer of the disc cover 32 and the lower layer of the disc cover 33, slots 36 for exposing the application area of the sample 39 of the measuring cell. Preferably, the test disk 31 is additionally provided with a registration slot 38 which may be located on the inner edge of the disk 31. During a measurement procedure, only the analyte test element, which is normally used for the determination of analytes, is exposed through the optical window, as shown in Fig. 14c. The analyte test disk 31 is able to rotate around its central point to bring a new analyte assay element into position as needed. By means of an analyte test disk it is possible to arrange a plurality of analyte test elements in a relatively small area. The same number of analyte test elements included in the analyte test strips would require a much larger area and, therefore, much more material, as illustrated, comparing the sizes of the analyte test disc and the analyte test strips in Fig. 14. While the unit area 40 of the analyte test disk 31 includes nine analyte test elements 41, the same area 42 would have only three analyte test strips. However, it is not possible to reduce the sizes of the test strips since the handling of smaller strips would become more difficult and would be less practical for the patient. Fig. 16a and Fig. 16b show the analyte test disk included in a meter where the sample application zone 39, 43b protrudes again from the meter housing. Not only for the analyte test strips but also for the analyte test disk it is possible to adapt the measuring device (analyte test system) to a left-handed and right-handed operation, as illustrated in Fig. 17. When a left-handed operating mode according to FIG. 17a is desired, the analyte test strip 57 is inserted into the meter from the underside, sample application area 43a, to receive the physiological or aqueous fluid protruding from the sample. Wrapped in the meter 58. At the end of the measurement, the analyte concentration is presented on the screen of the analyte test system 54. Similarly, a right-handed operating mode according to Fig. 17b can be performed by adapting the screen 54 of the analyte test system 59 to a reverse mode of operation by rotating the content presented on the screen 180a, which allows the insertion of the analyte test strip 57 into the meter from the upper side. Fig. 18 shows another possibility for disposing the analyte test elements in a way that saves space. In this embodiment, the analyte test elements are arranged side by side to form a tape 44 with a lateral extension to form the sample application zones 9. In the tape, the zone between two analyte test elements is provided. of a perforation or folding line 46 for separating a test element from an analyte used from the unused portion of the analyte test strip 44. By means of a zigzag fold along the perforation or folding lines 46 it is possible to construct a tape package for the analyte test device 48 that can be easily housed in a small container allowing easier distribution of the individual analyte test elements of the analyte test tape. Method for preparing the analyte test element The analyte assay element of the present invention, produced in the form of a disk or strip, can be easily prepared by processes similar to those normally used in the fields of printing, punching and lamination . The design of the analyte assay element allows for a simple and cost-effective production process, which is preferably, but not necessarily, continuous in nature. In a first step of the preparation method, a configuration of the sample distribution system is formed by creating zones of high and low surface energy on a substrate. In a preferred embodiment, the areas of high surface energy forming the access routes of the sample and the detection zones on the first and second surfaces are generated by the application of a hydrophilic formulation on a hydrophobic surface on a substrate. As detailed above, it is also possible to create zones of high and low surface energy by applying a configuration of the hydrophobic "guide elements" on a hydrophilic surface. In case the substrate has an intermediate hydrophobic character, it is also possible to print the hydrophilic access routes with the surrounding hydrophobic configuration. The substrate may be formed of a material such as glass, polyvinyl acetate, polymethyl methacrylate, polydimethylsiloxane, polyesters and polyester resins containing fluorene rings, polycarbonates and polycarbonate-polystyrene graft copolymers, modified terminal polycarbonates, polyolefins, cycloolefins and copolymers of cycloolefins and / or olefin-maleimide copolymers. The application of a hydrophilic configuration on a hydrophobic substrate and / or the application of hydrophobic "guide elements" on a hydrophilic substrate can be performed by flexography, lithography, gravure, solid ink coating methods or by a jet printing process. ink. However, the preferred method of manufacturing is flexography, which allows a high resolution printing along with a high speed production in presses and supports. It is an established technology for printing on polymeric film substrates and is widely used in the packaging industry. The optical detection process shown in Figure 8 requires a clear, clear ink with low viscosity for the hydrophilic configuration. Low viscosity inks are preferred to achieve a fine and uniform coating of about 2-4 microns. It is necessary that the optical window of the ink is in the wavelength range in which the indicator dye absorbs light after the chemical reaction. The requirements for hydrophobic inks, apart from their hydrophobic nature, are less stringent and could also be used to decorate the analyte test strip or disc with the desired color. The operation of a four-color flexo printing machine is an established practice and does not create operational problems. The same is applicable to the lithography device. Although UV-curing or solvent-based inks are applicable for preparing the analyte test element, electronic beam hardening (EB) inks are especially preferred. These inks provide greater resistance to mechanical and chemical factors and contain 100% polymers, optionally pigmented, but without volatile organic solvents or photoinitiators, which have been shown to affect the stability of the sensor chemicals. These performance characteristics with positive benefits derive from the ability of electrons to form cross-linked polymer films and penetrate the surface. The inks used in the hardening by EB are based on the polymerization capacity of the acrylic monomers and oligomers. Acrylic chemistry has a special importance in today's modern inks. (JT Kunjappu "The Emergence of Polyacrylates in Ink Chemistry," Ink World, February, 1999, p.40) The structure of the simplest acrylic compound, acrylic acid, is shown in formula (I) CH2 = CH-COOH (I) The double bond in the acrylic part develops during the interaction with the electrons (initiation) and forms a free radical that acts on other monomers forming a chain (propagation) that leads to high molecular weight polymers. As mentioned above, the radiation-induced polymerization does not require any external initiator since the radiation itself generates free radicals with the result that no initiating species will remain in the coating.

A variety of acrylic monomers are available for EB hardening, from simple acrylates such as 2-phenoxyethyl acrylate and isooctyl acrylate to prepolymers such as bisphenol A, epoxyacrylates and polyester / polyether acrylates (R. Golden J. Coatings Technol., 69 (1997), p. 83). This hardening technology allows the design of "functional inks" focused on the desired chemical and physical properties without the need for a solvent or the hardening systems required by the other inks, which can complicate the design process. Suitable hydrophobic inks will contain monomers, oligomers and prepolymers with hydrophobic functions such as isooctyl acrylates, dodecyl acrylates, styrene derivatives or partially fluorinated carbon chain systems. Inks with hydrophilic functions can be obtained from a wide selection of crosslinkable water-soluble polymers; useful are acrylate derivatives prepared from polyalcohols, polyethylene glycols, polyethylene oxides, vinylpyrrolidone, alkylphosphocholine derivatives and others; Particularly useful are organically modified silicone acrylates, which are a crosslinkable species of modified polysiloxane organ. Suitable coatings provide a contact angle with water typically less than 25a and a surface energy typically greater than 55 mN / m. The second step of the production process comprises the application of the catalytic formulation, which contains an enzyme or other compound which undergoes a catalytic or non-catalytic reaction with the analyte and, if necessary, a coenzyme, and an indicator dye, on the predetermined detection of the sample distribution system formed in the substrate providing the first surface, and the application of the calibration formulations containing different levels of the calibration compound or analyte to the predetermined detection zones of the sample distribution system formed in the substrate that provides the second surface. The accuracy of this deposition step is critical and defines the accuracy and performance of the analyte assay element. Preferably, both formulations are applied with the aid of high precision ink jet systems or piezoelectric print heads. The catalytic and calibration formulations must be prepared to be very soluble in the sample of physiological or aqueous fluid. Preferably, they are based on water. Therefore, these inks are composed mainly of water, enzymes, indicators or the appropriate calibration compound, and will dry at slightly elevated temperatures. The main aspect of these ink formulations is the rapid reconstitution of the chemical components after the application of the sample without compromising the hydrophobic zones of the analyte assay element. The next step comprises the rolling process, in which the base and coating layers presenting the first and second surfaces of the sample distribution system are laminated on a central layer, thereby defining a distance between the first and the second surface of the base and coating layers. The central layer provides a discontinuity by creating a cavity for the sample distribution system in the zones in which the sample distribution system is formed on the first and second surfaces of the base and coating layers. The high and low surface energy configurations formed on the first and second surfaces of the base and coating layers must be aligned to be basically congruent, allowing the formation of a functional system for distributing samples between the first and second surfaces. The precise x-register of the base and coating layers becomes a critical task for the function of the device; if this registration is not achieved, the sample distribution system will not work properly and / or will have a higher variability with respect to the specified volume of the sample. The registration tolerances should be at +/- 5% of the width of the hydrophilic access ways to allow good performance. The application of the central layer, which can be a double-sided adhesive tape with a preferred thickness of 80 microns, is less demanding due to the relatively large discontinuity in the material compared to the size of the hydrophilic access ways. Registration is especially important in continuous production lines when the substrate advances from several meters to tens of meters per minute. The expansion of the substrate and the tension of the coil make it more difficult to register in the x-direction (the direction of movement of the coil) than in the y-direction, perpendicular to the movement of the coil. An inventive method for the preparation of flexible polymer films, which provides an accurate record of the configurations of the first and second surfaces, is illustrated in Figures 19a-d, but shows the stages of a continuous process of coil production. In a first production step according to Fig. 19a, the configurations of the sample distribution system 6 of the base and coating layers are printed on a coil substrate 49, which represents the material of the element and the test strip analytes, respectively. As illustrated in Fig. 19a, the printed configurations of the sample distribution systems 6 are arranged on the substrates of the coil 49 in such a way that two sample distribution systems are opposed to each other and joined in the areas that later form the application areas of the sample. Thus, the position of the predetermined detection zones 6a, 6'a is fixed relative to one another and remains unaffected by the material expansion and the tension of the coil.

The dotted lines 50 indicate the future cut lines to separate the sample strips of analytes, while dotted lines 51 indicate the specular line of the strip example and the future fold line of the substrate in the roll. After printing the sample distribution zones, the detection zones 6a, 6'a of the sample distribution system are coated with the catalytic and calibration formulations. For example, the detection zones 6'a of the lower row of the coiled substrate 49, which will represent the first surface of the analyte test element, are coated with the catalytic formulation containing the enzyme and an indicator, while the zones 6a of the upper row of the coiled substrate 49, which will represent the second surface of the analyte test element, are coated with the calibration formulations containing different levels of the calibration compound; one of the calibration formulations (for example, placed in 6ai) does not contain the calibration compound and supplies the reading of the physiological or aqueous fluid in the detection step. Next, an additional layer is laminated to one of the surfaces, for example the surface 2a of the base layer 2, which represents the central layer 52 of the analyte test element as shown in Fig. 19b. The central layer 52 can be formed by an adhesive tape on both sides, which provides the interruptions 5 that expose the distribution systems of the sample 6 and which will create a cavity for the distribution systems of samples in the analyte test element afterwards. of the final assembly step. The analyte test element of the present invention is then assembled by folding two sides of rows along the fold line 51, for example with the help of a folding iron, as illustrated in Fig. 19c, creating a folded and rolled coil 53 as shown in Fig. 19d. Accordingly, a press roll can ensure a tight connection between the center layer and the base and cover layers. Finally, the laminated coil 53 is cut or perforated to the desired shape of the product, while the line 50 projects a typical form of the final analyte test strip on the coil 53 prior to the separation process. With the preparation method illustrated in Fig. 19d, the upper part of the substrate can be folded over the lower part without running the risk of losing registration in the x-direction of the coil and provides an easier method to obtain the registration. correct of the first and second surfaces that make up the sample distribution system compared to the single sheet process. The present invention provides an analyte test system that incorporates the means of quality control in a dry reagent test strip format that does not involve excessive demands on the strip production process, but eliminates the need for interventions of the user in the calibration and quality control procedures in combination with a close control of the performance of the strip at the time of the analysis of the samples.

Claims (36)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as novelty, and therefore the content of the following is claimed as property: CLAIMS 1. Analyte test element for determining the concentration of at least one analyte in a fluid of physiological or aqueous sample having a first surface (2a) and a second surface (4a) at a predetermined distance, opposite one with respect to the other, said both surfaces being provided with two substantially equivalent configurations forming high and low energy zones surface which are basically aligned in a congruent manner creating a sample distribution system (6) with at least two detection zones (6a), in which the applied physiological or aqueous fluid is substantially forced towards the high surface energy zones. An analyte test element according to claim 1, characterized in that the distance between the first and second surfaces is determined by a central layer (3) which is arranged between a base layer (2) and a covering layer (4) which they contain the first and second surfaces (2a, 4a). 3. An analyte test element according to claim 2, characterized in that the central layer (3) has a discontinuity (5) to form a hollow cavity together with the first and second surfaces (2a, 4a) of the base and coating layers (2). , 4), said hollow cavity being larger than the sample distribution system (6) formed by the high surface energy zones on the first and second surfaces (2a, 4a). The analyte test element according to one of the preceding claims, characterized in that said high surface energy zones are created by the application of crosslinkable and / or non-soluble hydrophilic and / or amphiphilic agents on the first and second surfaces (2a, 4a ). An analyte assay element according to claim 4, characterized in that said hydrophilic agents are selected from the group consisting of functionalized derivatives from polyalcohols, polyethylene glycols, polyethylene oxides, vinylpyrrolidones and organo modified polysiloxanes or copolymers of alkylphosphocholine and polyethylene glycol. 6. An analyte test element according to claim 1, characterized in that said first surface (2a) and second surface (4a) are hydrophobic and non-wettable by a physiological or aqueous fluid and are transparent to particular light in UV, IR near and / or the visible range of the electromagnetic spectrum. 7. Analyte test element according to one of the preceding claims, characterized in that said low surface energy areas are created by the application of a hydrophobic composition on the first and second surfaces (2a, 4a), said hydrophobic composition preventing the wetting of the area Coated by a physiological or aqueous fluid. An analyte assay element according to claim 7, characterized in that said hydrophobic composition contains isooctyl acrylates, dodecyl acrylates, styrene derivatives or systems with partially fluorinated carbon chains. 9. Analyte test element according to claim 7, characterized in that said first surface (2a) and second surface (4a) are hydrophilic and wettable for the physiological or aqueous fluid and transparent to particular light in UV, near IR and / or the visible range of the electromagnetic spectrum. An analyte assay element according to claim 9, characterized in that said first surface (2a) and second surface (4a) are rendered hydrophilic by physical or chemical vapor deposition of the hydrophilic compounds. 11. Analyte test element according to one of the preceding claims, characterized in that the base layer (2) and the cover layer (4) provided by the first and second surfaces (2a), 4a) are composed of a material selected from the group consisting of glass, polyvinyl acetate, polymethylmethacrylate, polydimethylsiloxane, polyesters and polyester resins containing fluorene rings, polycarbonates and polycarbonate-polystyrene graft copolymers, modified terminal polycarbonates, polyolefins , cycloolefins and cycloolefin copolymers and / or olefin-alemimi copolymers. 12. Analyte test element according to one of the preceding claims, characterized in that the n predetermined detection zones (6'a) of said first surface (2a) are coated with the catalytic formulation that promotes the detection of an analyte in a physiological or aqueous fluid, and the n predetermined detection zones (6a) of said second surface (4a) are coated by the n calibration formulations composed of m blank formulations and nm formulations with different levels of the calibration compound, where n is an integer greater than 2, m is an integer equal to or greater than 1 and n > m. 13. An analyte test element according to claim 12, characterized in that an additional detection zone (6c) is provided, which contains neither the catalytic compound nor the calibration compound, allowing the measurement of the background signals. The analyte test element according to claim 12 or claim 13, characterized in that said catalytic formulation covering the n predetermined detection zones (6'a) of the first surface (2a) allows the detection of a concentration of an analyte contained in a sample of physiological or aqueous fluid using transmission or absorbance photometry. The analyte assay element according to claim 12, characterized in that said calibration compound contained in the calibration formulation that covers the nm predetermined detection zones (6a) of the second surface (4a) is identical or substantially equivalent to the analyte and it is capable of inducing the same chemical reaction in the catalytic formulation as the analyte in the physiological or aqueous fluid sample. 16. An analyte assay element according to claim 15, characterized in that the calibration compound is glucose. 17. An analyte assay element according to claim 12, characterized in that the catalytic formulation contains, as reactive components, a promoter that undergoes a catalytic or non-catalytic reaction with the analyte and / or a coenzyme and an indicator that generates an optically detectable product. 18. An analyte assay element according to claim 17, characterized in that the promoter is an enzyme selected from the group consisting of dehydrogenases, kinases, oxidases, phosphatases, reductases and / or transferases. 19. An analyte assay element according to claim 18, characterized in that the promoter is a specific enzyme for glucose. An analyte assay element according to claim 17, characterized in that the indicator for determining the concentration of analytes is selected from the group consisting of aromatic amines, aromatic alcohols, azines, benzidines, hydrazones, aminoantipyrins, conjugated amines, conjugated alcohols and / or aromatic and aliphatic aldehydes. 21. Analyte test element according to at least one of claims 12 to 20, characterized in that the calibration formulation applied to the predetermined detection zones (6a) of the second surface (4a) contains an inert dye soluble in water in a predetermined and fixed ratio with respect to the calibration compound that allows a suitable reading device to evaluate the concentration of the calibration compound within the calibration formulation at a wavelength different from the wavelength used to measure the product of reaction of the catalytic formulation with the analyte. 22. An analyte assay element according to claim 21, characterized in that said inert water-soluble dye is selected from the group consisting of bright black BN; bright blue G; Carmoisine; coumarin 120, direct blue 2B; indigo carmine; cochineal red; ponceau 4R; rhodamine 19; sunset yellow; tartrazine; and / or a water soluble derivative of malachite green. 23. Analyte test element according to one of the preceding claims, characterized in that a sample application zone (9) is located at the end of a convex and lateral extension (10) on one side of said analyte test element. 24. Analyte assay arrangement including a plurality of devices according to one of the preceding claims, which are arranged symmetrically around a central point to form an analyte test disk (31) with application areas of outwardly facing samples (39). 25. An analyte test setup that includes a plurality of devices according to at least one of the preceding claims, which are arranged in a linear fashion to form an analyte test strip (44) with lateral extensions forming the application areas of the analyte. samples (9). 26. Method for preparing an analyte assay element comprising the steps of: applying zones of high and low surface energy on a base layer (2) having a first surface (2a), the areas of high surface energy forming a hydrophilic access path with n predetermined detection zones (6'a), where n is an integer equal to or greater than 2, apply a corresponding configuration of high and low zones surface energy on a coating layer (4) having a second surface (4a), applying a catalytic formulation on the n detection zones (6'a) of the first surface (2a), said catalytic formulation promoting the detection of a concentration of analytes contained in a sample of physiological or aqueous fluid using transmission photometry or absorbance, applying the n calibration formulations on the n detection zones (6a) of the second surface (4a), constituted said n calibration formulations of m blank formulations and n- formulations with different levels of the calibration compound, where m is an integer of at least 1 and n > m, which is identical or substantially equivalent to the analyte and is capable of causing the same chemical reaction in the catalytic formulation as the analyte in the physiological or aqueous fluid sample, laminating the layers of the first and second surfaces at the opposite locations of a central layer (3) having a discontinuity (5) that provides a cavity for the sample distribution system (6) formed by the high surface energy zones on the first and second surfaces of the base and coating layers, drilling or Cut the laminated sheets into their final shape. 27. Method for preparing an analyte test element according to claim 26, characterized in that said high surface energy zones are created by the application of crosslinkable and / or non-soluble hydrophilic and / or amphiphilic agents on the first and second surfaces. 28. Method for preparing an analyte test element according to claim 27, characterized in that said high energy surface areas are printed on the first and second surface by means of flexography, lithography, gravure, solid ink coating methods or jet printing. ink. 29. Method for preparing an analyte test element according to one of claims 26 to 28, characterized in that said areas of low surface energy are created by the application of the hydrophobic compound on the first and second surfaces, said hydrophobic compound preventing the humidification of the area covered by a physiological or aqueous fluid. 30. Method for preparing an analyte test element according to claim 29, characterized in that said first and second surfaces are rendered hydrophilic by physical or chemical vapor deposition of the hydrophilic compounds. 31. Method for preparing an analyte test element according to one of claims 26 to 30, characterized in that the high surface energy zones of the first and second surfaces are physically raised from the low surface energy areas by etching or embossing. 32. Method for preparing an analyte test element according to one of claims 29 to 31, characterized in that said areas of low surface energy are printed on the first and second surfaces by means of flexography or lithography. 33. Method for preparing an analyte test element according to one of claims 26 to 32, characterized in that the base layer (2) and the cover layer (4) are formed from a flexible substrate (49) and folded along a centered longitudinal fold line (51) to include the central layer (3) in such a way that the sample distribution system (6) with the predetermined detection zones (6'a, 6a) of said first surface (2a) and second surface (4a) are aligned and registered to be mainly congruent. 34. An analyte test system for determining the concentration of an analyte in a physiological or aqueous sample fluid comprising an analyte test element or an analyte assay arrangement according to one of claims 1 to 25, characterized in that the predetermined detection zones (6'a) of a first surface (2a) are coated with a catalytic formulation that favors the detection of an analyte in a physiological or aqueous fluid, and the n predetermined detection zones (6a) of a second surface (4a) are coated with n calibration formulations composed of m blank formulations and nm formulations with different levels of the calibration compound, where n is an integer greater than 2, m is an equal whole number or greater than 1 yn > m, the detection means for detecting absorbance changes of the light of the physiological or aqueous sample located in the predetermined detection zones and for obtaining results from 2n predetermined detection zones, and the processing means for calculating The nm calibration coefficients of a polynomial calibration equation that obeys and a regression coefficient to validate the quality of the n-m calibration coefficients calculated from the calibration equation. 35. Method for determining the concentration of at least one analyte in a physiological or aqueous sample, said method comprising applying a physiological or aqueous sample to an analyte test element having a first surface (2a) and a second surface (4a) at a predetermined distance opposed from each other, said two surfaces being provided with two substantially equivalent configurations forming high and low surface energy zones that are aligned mainly congruently to create a sample distribution system (6) with at least two detection zones, in which the applied physiological or aqueous fluid is forced towards the areas with high surface energy, the detection of the signals produced in the different detection zones, and the relationship of the signals to determine the amount of analyte (s) ) in the physiological or aqueous sample. 36. Analyte test element for determining the concentration of at least one analyte in a fluid of a physiological or aqueous sample having a first surface and a second surface at a predetermined distance opposite from each other, characterized in that one of the surfaces first and second is provided with a hydrophilic / hydrophobic configuration and the corresponding surface provides a homogeneous configuration of hydrophilic pixels surrounded by a hydrophobic zone which, therefore, creates above all a semi-hydrophilic and semi-hydrophobic surface, by means of which Hydrophilic and semi-hydrophilic zones create a sample distribution system with at least two detection zones. KESUMEN OF THE INVENTION The invention provides a device for determining the concentration of an analyte such as glucose, cholesterol, free fatty acids, triglycerides, proteins, ketones, phenylalanine or enzymes, in a physiological fluid such as blood, serum, plasma, saliva, urine , interstitial and / or intracellular fluid, the device has an integrated calibration and an appropriate quality control system for dry reagent test strips with a very small sample volume of approximately 0.5 μL based on a new sample distribution system. The production of the test element of the inventive analyte involves only a small number of uncomplicated production steps that enable the cheap production of the strips.