RU2398235C2 - Test-system with application of elements of non-enzymatic recognition of analysed substance - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: test-element has surfaces located at set distance opposite each other and containing two similar structures that form sections with high and low surface energy. At least one of sensitive sections of the first and second surfaces is provided with at least one element of non-enzymatic recognition able to provide reaction of affinity with analysed substance. Method of test-element manufacturing includes formation of sections with high and low surface energy on bottom layer with the first surface. Sections with high surface energy form hydrophilic system of sample distribution, formation of structure of sections with high and low surface energy on upper layer with the second surface, covering given sensitive sections of the first surface with calibration compositions, covering given sensitive sections of the second surface with recognition composition, which contains recognition element, and application of layers of the first and second surfaces onto opposite sides of central layer with a cut. Described is test-system which includes test-element by i.i. 1-15, and method of qualitative and/or quantitative determination of at least one analysed substance in a sample of physiological or water liquid, using application of physiological sample liquid on test-element by i.i. 1-15.

EFFECT: increase of analysis accuracy.

26 cl, 21 dwg, 2 tbl

Description

FIELD OF THE INVENTION

The present invention relates to a test system for the quantitative and qualitative determination of an analyte in a liquid medium, such as a sample of an aqueous or physiological fluid. In a preferred embodiment, the present invention relates to a test system for quantitatively and qualitatively evaluating a reaction between a host molecule and a guest molecule, in particular for affinity studies and immunoassays.

BACKGROUND OF THE INVENTION

Diagnosis and confirmation of various diseases and health conditions from the very beginning was the main task for specialists in medicine. Currently, doctors have at their disposal many diagnostic methods that make it possible to accurately diagnose and confirm various diseases and conditions of patients in body fluids. Determining the various conditions of a patient, in particular, is especially important for doctors, since it is necessary to meet different needs and requirements. The determination of various analytes in body fluids, such as blood, serum and urine, plays a significant role in the fight against various diseases.

In addition, doctors and patients require accurate test result systems and products that are easy to use and convenient to use, especially for monitoring and home care. This convenience is closely related to the number of operations performed by the doctor or patient to obtain the final test results and the required sample volume when the test system requires blood or serum as a sample liquid and a non-invasive sampling procedure.

Typical examples are registration of human chorionic gonadotropin during pregnancy tests or determination of cardiac troponin I, cardiac troponin T, SCMB, myoglobin and natriuretic brain peptide (BNP) as cardiac markers for determining actual heart attack.

Additional examples of analytes that are increasing in importance are tumor markers that are used and crucial for monitoring patients after therapy to describe the course of the disease before any carcinogenic tissue can be detected clinically or by imaging. Examples of these markers are CEA, PSA, CA19-9, CA125. CEA or carcinoembryonic antigen, a blood-borne protein, first mentioned as being produced by tumors of the gastrointestinal tract.

Similarly, another example of increasing importance in the treatment and / or control of a disease synonymous with complications related to diabetes is diabetic retinopathy. This condition is a retinal disease and, according to many reports, occurs in about a third of patients with diabetes.

PSA, or a specific antigen of the prostate, is produced by the normal prostate. This is an enzyme called serine protease, which usually acts as an anticoagulant to preserve seminal fluid. Usually only small amounts enter the circulatory system. An enlarged prostate and a malignant prostate secrete significant amounts into the circulatory system and urine, which can be detected by diagnostic and screening methods.

All of the tests listed above and many others use affinity reactions of the analyte of interest (guest molecule) and a recognition element specific for the analyte (host molecule) to quantify the presence or absence of, respectively, the quantitative assessment of the concentration of analyte molecules.

Specialists in this field classify the analysis as an immunological analysis. In the case of the affinity reaction described above in the examples, carried out between an antibody or antibody fragment as a recognition element, the analyte of interest is then usually called an antigen. This type of affinity study is most widely known and used in the field of clinical and therapeutic diagnostics.

In 2003, 9.6 million US citizens were recognized as having cancer. A study by the American Cancer Society indicates that early recognition and screening can reduce mortality from cancer, such as colorectal cancer by 30%, cervical cancer by 60%. In addition, other types of diseases that are spreading more and more in the West include, among others, obesity, diabetes, and arthritis. In addition, the diagnosis of diseases using immunological tests will give doctors the possibility of early use of drugs and therapy to reduce the likelihood of complex and often invasive therapy. Such therapy can be dangerous due to the risk of bacterial infection, which is expensive, and the probability of the patient recovering completely within a certain time after the manifestation of the disease in the patient's body is reduced.

In particular, while the spread of some viruses worldwide has declined, the spread of other viruses has increased, and the World Health Organization reports that by 2005 the estimated number of people with immunodeficiency virus (HIV) in the world will reach 40 million - an increase of 8% relative to the year 2000. Additional data suggests that third-world countries or developing countries show a significant increase in the number of people diagnosed with this disease, and for some countries there is often an increase of 15% compared to previous comparison dates.

Therefore, an urgent problem for doctors is their inability to quickly and accurately obtain test results to determine if a patient is suffering from any disease. One of the protocols for testing diseases such as HIV, tuberculosis, and cancer is to take a blood sample from a patient and transfer it to a laboratory for analysis. Indeed, such a protocol can be problematic for the patient, since the time delay between the initial consultation and the receipt of the test result causes mutual stress and unnecessary delay in treatment and, therefore, an increased risk for patients. However, on-site diagnostics and clinics that accept patients without an appointment are becoming more common in some countries, balancing the apparent inefficiency of the health departments, but the prices are attractive only to the most affluent members of society.

In addition, the equipment for tests in the doctor’s office may be expensive for some practitioners, and some may prefer to send fluid samples to the laboratory for analysis. Additionally, the cost of seeking medical care in some countries when visiting a doctor may be excessively high for some people, since not all insurance cases are free for on-site diagnostics. In addition, the diagnosis and monitoring of the disease in some cases can be carried out twice, i.e. doctors require confirmation of the presence or absence of the disease in the body fluid, and, secondly, the degree of development of the disease by analyzing the level of concentration of the analyte in the body fluid.

Thus, people who feel they are at risk of disease benefit from on-site diagnostic tests (PoC) or home-based diagnostic tests, since the latter allows the test to be carried out in an environment that is convenient for them. In addition, doctors achieve effective daily work if they can perform a diagnostic test at the workplace without resorting to expensive laboratory services. In many cases, providing such diagnostic kits gives the patient an element of control of his disease.

There are various test elements for either testing at home physiological conditions such as diabetes, pregnancy and fertility, but there is minimal test equipment that can provide a simple diagnostic tool to obtain information about the condition and course of the disease with quick and accurate results when diagnosing place or at home.

There are various principles of registration (detection) that are applicable in equipment for laboratory tests and (or) therapeutic test elements that determine the presence and level of an analyte in a test sample, for example, immunological analysis of agglutination, immunological analysis of diffusion, analysis of an enzyme modulator, immunological tests for fluorescence-based and coenzyme-apoenzyme analysis.

Accordingly, many modern industries and, in particular, the field of metabolism monitoring, face the problem of providing a test element with the following characteristics and properties:

- the test element must provide either accurate qualitative or accurate quantitative test results in aqueous fluids or body fluids with as few steps as possible for the user;

- only a small amount of sample should be needed for the test element, so the doctor or patient can use minimally invasive methods of sampling;

- the test element should provide only a small number of technological operations during production and, therefore, inexpensive manufacturing, and be used for products that help patients with self-monitoring of physiological parameters, and (or) the doctor at the place of work.

The prior art describes systems and examples of solutions or partial solutions to the above problems, in particular the publications mentioned below are particularly interesting for the described invention.

US Pat. No. 4,213,893 discloses a flavin adenine dinucleotide (FAD), a labeled conjugate, used as a labeled conjugate in specific binding assays to determine a ligand or a specific member of a binding pair in a liquid medium such as serum. FAD labeled conjugates used in ligand binding assays are monitored for analysis by measuring FAD activity, i.e. activity of a coenzyme or prostatic group of a labeled conjugate.

US Pat. No. 4,708,933 describes an analysis of a homogeneous solid-state immunoliposome, which uses the separation of the lateral phase of an antigenic liposome, leading to impaired stability and lysis of the liposome, which can be quantified and used to determine the presence and (or) concentration of antigens, antibodies and similar reagents in biological fluids .

In an article by RJ Tyhach et al. In Clinical Chemistry 27: 1499-1504; "Adaptation of Prostatic-Group-Label Homogeneous Immunoassay to reagent - strip format", published in 1981, describes a method for immunological analysis of the prostatic group label (PGLIA), carried out in the form of a strip with a reagent, using flavins N6- (N′- 2,4-dinitrophenyl-6-aminohexyl) adenine dinucleotide (DNP-FAD) as a derivative of the prosthetic group and 6-N- (2,4-dinitrophenyl) aminocaproic acid (DNP-caproate) as a competitive ligand. Non-antibody DNP-FAD combines with the glucose oxidase apoenzyme, which reacts with glucose and oxygen, creating color through a peroxidase-linked system. The rate of color formation, therefore, depends on the concentration of DNP caproate.

In an article by Butler et al. In Journal of Clinical Investigation 115: 86-93 (2005); "SDF-1 is both necessary and sufficient to promote proliferative retinopathy," published in 2005, describes the relationship between a protein known as SDF-1 and retinopathy.

EP 163393 describes a method for immunological analysis of latex to determine the presence of a ligand of interest in an undetectable body fluid and the presence of a chemical additive containing a halogen-substituted carboxylic acid or a salt of this acid in the reaction mixture to reduce the non-specific effect on the immunological analysis of latex. The addition of such an additive allows the use of immunological agglutination assays to quantify the levels of endogenous metabolites and to control specific ligands of interest, such as drugs, therapeutic reagents, and specific binding proteins in the samples.

WO 2002/086472 describes the use of fluorescent molecular rotors in which fluorescence intensity changes based on the viscosity of the medium. Molecular rotors are modified with hydrocarbon chains or hydrophilic groups to allow measurement of the membrane or fluid viscosity.

PCT / EP 2004002284 describes a test system for photometric detection and quantification of an analyte, for example glucose, in a physiological fluid, such as blood, which is equipped with an integrated calibration system using the standard addition method. This element contains the first and second surfaces forming a sample distribution system, the sensitive sections of which are coated with a catalytic and calibration composition, respectively, which allows the detection / determination of the analyte by enzymatic reaction.

However, to date there is no test system suitable for on-site or at-home diagnostics that would allow evaluating the non-enzymatic reaction between the analyte of interest and the recognition element specific to that analyte when performing built-in calibration and (or) measuring quality control for the analyte of interest substances in a given sample of physiological or aqueous fluid.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a test element for determining the concentration of at least one analyte in a sample of physiological or aqueous fluid with a first surface and a second surface at a predetermined distance from each other, both surfaces being provided with substantially the same structures forming sections with high and low surface energy, which are aligned (aligned), essentially congruent, so that areas with high surface energy form a distribution system dividing the sample with at least two sensitive areas, where at least one of the sensitive areas of the first and second surfaces is provided with at least one non-enzymatic recognition element of the analyte.

In a preferred embodiment, the test element has n predetermined sensitive areas (6a) of the first surface (2a), covered with n calibration compounds (18) made of m control (“blank”) compositions and nm compositions containing different levels of the calibration compound (33 ), while n is an integer greater than 2, m is an integer equal to or greater than 1, and n> m, and

n given sensitive areas (6′a) of the second surface (4a) coated with a composition containing a specific element (32) of non-enzymatic recognition of the analyte.

The present invention also provides a test system consisting of a measuring device and a test element, for example, in the form of a strip, for non-enzymic affinity reactions between the analyte of interest and a specific non-enzymatic recognition element of the analyte. For built-in calibration and quality control mechanisms, the test system uses standard adding methods using a test element sample distribution system containing the required standards and (or) quality control. The system is optimized for immunological, receptor, or other affinity studies in a quick and easy way with a simple and one-time test element, preferred for on-site diagnostics and home testing.

The manufacture of the test element of the present invention does not require a large number of technologically complex operations and ensures the production of inexpensive elements.

For a better understanding of the features and advantages of the present invention, the following is a detailed description of examples and preferred embodiments in combination with the attached drawings:

Brief Description of the Drawings

1 is a perspective view of one embodiment of a test element of the present invention in the form of a test strip;

FIG. 2 is a perspective view of the embodiment of FIG. 1, showing an enlarged scale sampling section and a sample distribution system;

figure 3 shows a perspective view in parts of the element of figure 1, showing three separate layers of the structure;

figure 4 shows various forms of a cutout of the Central layer, forming a cavity for the sample together with the first and second layer;

5 shows a cross-sectional view of a sensitive portion of a sample distribution system constructed with hydrophobic guide elements;

FIG. 6 is a cross-sectional view of another embodiment of a sensitive portion of a sample distribution system using hydrophilic channels;

Fig. 7 shows a schematic principle of an agglutination reaction: A) introducing a fluid sample medium, B) dissolving the recognition and calibration composition, C) final agglutinate formation between the analyte, the recognition element and the calibration compound;

Fig. 8 shows a schematic principle of agglutinating microparticles: A) introducing a fluid sample medium, B) dissolving the recognition and calibration composition, C) final agglutinate formation between the analyte, the recognition element immobilized on microparticles and the calibration compound;

Fig. 9 shows various embodiments of a sample distribution system with various channel designs and sensitive areas suitable for various calibration methods;

figure 10 shows the sample-sensitive portion of the sample distribution system of figure 6 in combination with a light emitter and a recording (detection device in cross section, configured to measure absorption;

figure 11 shows the location of the recording (detector) device for a test element configured to evaluate light scattering or fluorescence;

on Fig schematically shows the results and assessment for the quantitative analysis of non-competitive A) and competitive analysis B);

in Fig.13 shows examples of determining the concentration of the analyte using the method of linear addition of the standard;

Fig. 14 is a graph showing a validation method for a calculated result and calibration data;

on Fig shows examples of fluorescent dyes with non-specific affinity of proteins;

on Fig shows a schematic principle of the agglutination reaction by means of a fluorescent molecular rotor: A) the introduction of a liquid sample medium, B) the dissolution of the recognition and calibration composition, C) the final formation of agglutinate between the analyte, recognition element, calibration compound and a non-specific fluorescent molecular rotor as reporter element;

on Fig shows examples of non-specific fluorescent molecular rotors;

Fig. 18 shows a schematic principle of an affinity reaction using a specific fluorescent dye as a reporter element: A) introducing a fluid sample medium; B) dissolution of the recognition and calibration composition, C) the final formation of an affinity complex between the analyte, recognition element, calibration compound and a specific reporter element;

on Fig shows the principle of the reaction of recognition analysis using apoenzyme (AARA from the English. "apo-enzyme assisted recognition assay");

on Fig shows the effect of registration failures during lamination on the sample volume of the test element, as well as the upper part relative to the cross-sectional view of an alternative embodiment (c), which provides large allowable limits for fixing the lower and upper layers without compromising the quality of the test strips;

on Fig shows a method of manufacturing a test element used for affinity analyzes and immunological analyzes.

DETAILED DESCRIPTION OF THE INVENTION

As shown in FIGS. 1 and 2, the test element 1 for analyzing a substance of the present invention is a multilayer arrangement comprising a lower layer 2, a central layer 3 located above the lower layer 2, and an upper layer 4 located above the central layer 3. The central layer 3 contains a cutout 5, which forms a cavity in combination with the lower layer 2 and the upper layer 4. A sample distribution system 6 is located inside the cavity, which communicates with the sampling section 9 located on the edge of the test strip. The sampling portion 9 used by the user is preferably in the form of a rounded protrusion 10 located on one of the main sides of the test strip for the analyte for easier sampling. On the other main side of the test strip against the sampling section 9, 10, there is a vent 11 through which the filling system of the distribution of the physiological or water-based liquid displaces the air and enters the desired sensitive sections 6a, 6a (see FIG. 3). It should be noted that with this design, only one ventilation hole is required, regardless of the number of specified sensitive sections used inside the test element. The described elements of the sample distribution system with high surface energy areas, a sampling area, a ventilation hole, a central layer and a cutout in the central layer form the test element itself, which creates an internal capillary effect, which ensures the distribution of physiological or aqueous liquids to the given sensitive areas.

In addition, the test strip 1 contains recording (positioning) elements 7, 8, used to distinguish several types of test elements of the analyte to determine the various analytes. By means of this, the measuring device for several analytes is configured to carry out a special program or procedures with selectable parameters when a strip is introduced to determine the specific analyte.

As shown in FIG. 3, in which the multilayer structure of FIGS. 1 and 2 is shown in a disassembled form, the lower layer 2 provides a first surface 2a, and the upper layer 4 provides a second surface 4a. The first surface 2a and the second surface 4a are constructed with portions that create a sample distribution system 6. The structure of the sample distribution system 6 contains a predetermined number of analyte-sensitive sections 6a and sample channels 6b, which are aligned and fixed mainly congruently during assembly of the multilayer structure. The central layer 3 provides a distance between the first surface 2a of the lower layer 2 and the second surface 4a of the upper layer 4 and contains a cutout 5 for creating an internal cavity together with the first surface 2a of the lower layer 2 and the second surface 4a of the upper layer 4. The sample distribution system 6, which is formed between the first surface 2a and the second surface 4a, it is located inside the cavity created by the cutout 5 of the central layer 3 and the first surface 2a of the lower layer 2 and the second surface 4a of the upper layer 4. Preferably, the inner the cavity is essentially structurally larger than the sample distribution system.

Since the purpose of the cutout 5 of the Central layer is only to create a cavity for the system 6 of the distribution of the sample, the cutout 5 of the Central layer 3 may have a different shape; examples are shown in figure 4. On figa shows the cavity 12 of the test element in the shape of an umbrella, on fig.4b shows the rectangular cavity 13 of the test element, and on figs - cavity 14 for the sample has a circular shape. The cutout 5 of the central layer 3 does not affect the size of the given sensitive sections 6a and the size of the channels 6b of the sample distribution system 6 and, therefore, does not affect or does not change the required sample volume. Compared to the sample distribution system 6, the cavity shape shown in FIG. 4 is quite simple, thereby providing the use of simple punch dies and quick processing with less installation accuracy requirements.

The sample distribution system 6, located in the cavity formed by the cutout 5 of the central layer 3 and the first surface 2a of the lower layer 2 and the second surface 4a of the upper layer 4, is formed by creating areas with high and low surface energy on the indicated surfaces 2a and 4a. The high and low surface energy portions on the first surface 2a of the lower layer 2 and the second surface 4a of the upper layer 4 are aligned and fixed substantially congruently with respect to each other. Since the applied physiological fluid or any other water-based sample moistens only areas with high surface energy, it is thus limited within the specified flow channels 6b and sensitive sections 6a of the sample distribution system 6 and between the first surface 2a of the lower layer 2 and the second surface 4a top layer 4.

Figure 5 shows the design of a sample distribution system using hydrophobic "guide elements". In this embodiment, the test elements of the analyte of the present invention - the lower layer 2 and the upper layer 4 are covered with a hydrophobic layer 16, with the exception of areas that form the channels of the sample, and sensitive areas. The hydrophobic layer 16 creates a low surface energy region that exerts a repulsive force with respect to the applied sample liquid 15 and, therefore, limits the sample liquid 15 to the high surface energy regions that form the sample distribution system.

Preferably, the hydrophobic layer 16 is applied to the hydrophilic surface 2a, 4a, which is wetted with physiological fluid or fluid 15 of an aqueous sample. The procedure described above requires a hydrophilic surface that can be made from a natural hydrophilic polymer, such as cellophane or glass, as well as based on hydrophobic surfaces of common polymers (examples are given below), giving the hydrophobic surface hydrophilic properties using a coating or physical or chemical plasma deposition hydrophilic monomers that can be evaporated in vacuo, for example, silicon dioxide, ethylene oxide, ethylene glycol, pyrrole or acrylic acid. Therefore, the structure of the "guide elements" can be realized by printing hydrophobic inks on the hydrophilic surfaces of the lower and upper layers.

A suitable hydrophobic paint has a contact angle with water usually greater than 100 ° and surface energy usually less than 25 mN / m and usually contains monomers, oligomers and polymers with hydrophobic properties, imparting hydrophobic properties to the additive or hydrophobic pigments and fillers.

6 shows another design of a sample distribution system using hydrophilic channels. In this embodiment of the test element, the natural hydrophobic lower layer 2 and the upper layer 4 are coated with hydrophilic compounds or varnish 17, whereby areas with high surface energy are formed.

The hydrophilic reagent 17, which is printed onto the hydrophobic surface 2a, 4a, is strongly wetted by a physiological or aqueous sample liquid 15; thus, areas with high surface energy that create hydrophilic channels in the sample distribution system exert a positive capillary force on the applied physiological or water-based sample liquid to transfer sample liquid to the individual sensitive areas.

The hydrophilic structure can be realized by printing hydrophilic or amphiphilic reagents on a hydrophobic surface. Paints with hydrophilic properties can be selected from a wide range of high molecular weight polymers and mixtures thereof, soluble in polar solvents such as water or alcohols. Particularly suitable are derivatives of alginate, cellulose and cellulose ester, hydroxyethyl cellulose, resins, acrylic acid, polyvinyl alcohol, polyethylene glycol, polyethylene oxide, polyvinyl pyrrolidone, polystyrene sulfonate, poly (methyl vinyl ether / maleic acid) trimethyl vinyl methyl copolymers -phosphocholine. Additional optimization can be achieved through the use of organically modified additives of silicon acrylates, which are a cross-linked type of organo-modified polysiloxanes and fluorinated surfactants. Suitable coatings provide an angle of contact with water typically less than 35 ° and surface energy typically more than 50 mN / m.

The lower layer 2 and the upper layer 4, suitable as a substrate for printing, can be formed from a material such as glass, polyvinyl acetate, polymethylmethacrylate, polydimethylsiloxane, polyesters and polyester resins containing fluorene rings, polystyrenes, polycarbonates and grafted copolymers of polycarbonate-polystyrene, terminal modified polycarbonates, polyolefins, cycloolefins and copolymers of cycloolefins and (or) copolymers of olefin-maleimide.

In the case where the substrate is of the intermediate hydrophobic type, it is also possible to print hydrophilic channels with a surrounding hydrophobic structure, i.e., a combination of the structures of FIG. 5 and FIG. 6.

In an alternative embodiment, either the first or second surface is provided with a hydrophilic / hydrophobic structure (6, 16), since the corresponding surface provides a homogeneous structure of hydrophilic pixels surrounded by a hydrophobic region, thereby creating a surface with semi-hydrophilic, semi-hydrophobic properties (amphiphilic surface nature), which eliminates the need to combine the hydrophilic and hydrophobic structures (6, 16) of the first surface with the equivalent hydrophilic and hydrophobic structure ( 6 ′, 16 ′) of the second surface. The properties of such an amphiphilic surface can be easily designed through the geometric structure of the hydrophilic pixels and the general relationship between the hydrophilic and hydrophobic region. In the described invention, the amphiphilic character, respectively, the ratio between the hydrophilic pixels and the hydrophobic regions is designed so that the sample fluid passes from the hydrophilic pixel to the hydrophilic pixel only if the opposite surface provides a hydrophilic character. If the opposite surface provides a hydrophobic character, the movement of fluid inside the capillary gap of the test element is stopped. This mechanism allows the creation of a functional test element according to the above method without the strict requirement of accurate registration of the corresponding structure of the sample distribution system provided on the first and second surfaces.

However, it is preferable that both the first and second surfaces be provided with the same structures with high and low surface energy to ensure rapid distribution of sample fluid within the hydrophilic channels of the sample distribution system.

Moreover, it is possible to physically lift areas with high surface energy of the first and second surfaces relative to areas with low surface energy by etching, embossing, or simply printing the hydrophilic layer 17 with a thickness of about three to five times more on the first and second surfaces. Due to this rise, the capillary gap of the hydrophilic channels becomes smaller relative to the surrounding area and has a stronger effect of the capillaries on the sample liquid.

The volume requirement for the sample distribution system contained in the analyte test element of the preferred embodiment is very low and is about 0.5 μl - 1.0 μl, only about 100 nl - 150 nl per sensitive area is required, depending on whether areas with high and low surface energy are created by hydrophobic guide elements or hydrophilic channels or a combination thereof. However, for specialists it is obvious that the volume of the sample distribution system is different for different designs and depending on the number of used given sensitive sections.

As shown in FIGS. 5 and 6, the areas with high surface energy of the first and second surfaces 2a, 4a, forming sensitive areas coated with compositions 18, 19, facilitate registration and allow the analyte to be determined in the sample liquid 15. In the embodiment shown in FIGS. 5 and 6, the first surface 2a of the sensitive portion is coated with a calibration composition 18. The calibration composition comprises a calibration compound, which may be an analyte or a molecule equivalent to the analyte that has affinity for the recognition element. The second surface 4a of the sensitive portion is coated with a recognition composition 19 containing a recognition element, for example, a receptor, an antibody or an antibody fragment, such as a Fab fragment, which has affinity for the analyte and which can facilitate the registration and determination of the analyte in the sample fluid. Specific examples of the calibration compound as well as recognition elements are given below.

As mentioned above, depending on the need for the doctor to perform the test, various diseases of the body require different sensitivity and different dynamic ranges, i.e. HIV test systems require a highly sensitive approach for detecting a threshold, but not a wide dynamic range, while the detection of glycosylated hemoglobin HbAlc, a 3-month marker for the development of diabetes and patient compliance, requires a wide dynamic range, but not a very sensitive threshold.

Further, the occurrence of diabetes complications, such as diabetic retinopathy, requires a very sensitive approach using a protein-based recording agent. For example, the detection of such a complication can be done with at least double examination, i.e., the occurrence of retinopathy is usually monitored by ophthalmologists, and the diagnosis and condition of diabetic retinopathy is performed according to a detailed retinal examination with an ophthalmoscope. In the second method for detecting diabetic retinopathy, it is known that the concentration of Factor-1 protein based on stromal cells (SDF-1) increases with the development of diabetic retinopathy, correlating with the severity of the disease. Such a protein, therefore, can be used as a basis for analysis in the registration and monitoring of complications of a disease, such as retinopathy.

Thus, registration methods and modes should be adapted to the requirements of diagnostic needs according to the requirements of patient therapy. The examples described below are preferred not only as an implementation of a test element to perform the required diagnostic task. They contain most cases of chemical and (or) biochemical compounds, such as fluorescent dyes, microparticles, coenzymes and apoenzymes, providing or enhancing the registration of the desired affinity reaction (recognition reaction) between the analyte and the corresponding analyte specific recognition element, thus, you can apply mainly to these compounds as reporter elements or to their many as a reporter system.

Test systems using reporter elements for affinity analysis

Agglutination Methods

7 shows a diagram and principle of a first embodiment of the present invention, applicable for affinity analysis and immunological analyzes, or generally established for recognition studies. In this embodiment, an agglutination reaction is described that indicates qualitatively a quantitative reaction between the analyte of interest 31 and its specific recognition element 32. In this particular case, analyte 31 may be a protein, such as a virus protein or hCG choriogonadotropin, and recognition element 32 may be an antibody for that protein.

Fig. 7 schematically shows this reaction if a liquid sample 15 containing the analyte of interest 31 enters a sample-sensitive region containing a recognition element 32 and a calibration connection 33 and attached to the first and second surfaces 2a and 4a. The recognition element 32, as well as the calibration compound 33, are dissolved by the sample liquid 15 and the recognition element 32, which reacts with the calibration compound 33, as well as with the analyte 31 contained in the sample liquid. For the course of the reaction, the recognition element 32 is capable and must bind to several molecules of the analyte, thus, the recognition elements 32, therefore, crosslink or agglutinate all the molecules of the analyte into large clusters 34. The size of these clusters or particles leads to the scattering of laser light depending on their physical size. If the clusters are smaller than the wavelength of light, the cluster causes a special type of scattering, called Rayleigh specialists in this field, since the clusters are larger than the wavelength of light, cause scattering called Mie scattering. Various types of scattering can be used to determine the presence, or quantity, of the analyte, and the scattering pattern is different depending on the laser wavelength.

In the case where the recognition element or elements 32 are immobilized on microparticles 35, for example, of gold, latex or polystyrene, as shown in Fig. 8, the agglutination reaction between a plurality of such microparticles with increased sensitivity and the analyte can be monitored using a simple photo-measuring device or even eyes. During the agglutination reaction, the number of single microparticles decreases due to agglutination, thus, the number of scattering centers in the sample is reduced, and the light is scattered after the described reaction, providing a higher light transmission through the sample-sensitive area.

Similarly, the absence of an affinity reaction between the analyte and the recognition element in the liquid medium of the sample is detected by the absence of an agglutination reaction and, therefore, is characterized by a low light transmission through the sample-sensitive region.

The test element is provided with a coating of sensitive sections 6′a of the first surface 2a of the lower layer 2 with a suitable recognition element 32 relative to microparticles 35 with increased sensitivity. Preferably, the microparticles 35 may be of latex, gold or gelatin, but more preferably of gold. Other known particles can be used, such as carbon, polystyrene or the like, which is obvious to those skilled in the art. Alternatively, the microparticles 35 can be coated on the sensitive areas 6a of the second surface 4a of the upper layer 4. In addition, the microparticles can be coated on both the sensitive areas of the first surface 2a and the sensitive areas of the second surface 4a, since the analyte is not analyzed calibration composition 18 is required relative to calibration compound 33.

In other and more preferred embodiments, the sensitive sections 6a of the first surface 2a are coated with calibration formulations 18, which allow the analysis of positive and negative control to be performed in parallel with the determination of the analyte, thereby creating a guarantee for the user that the measurement is correct and that the test system, and the test element used is also fully functional. Such a feed through a distribution system provides a high concentration of the calibration compound (analyte or molecule equivalent to the analyte, which have an affinity for the recognition element) in one sensitive area, for example 6′a3, which allows the agglutination reaction to be completely carried out. Thus, the readings of the sensitive section 6′a3 correspond to approximately 100% or the full scale of the readings of the measuring device. The second sensitive portion, for example 6′a2, contains an inhibitor compound that suppresses the affinity reaction of the analyte 31 with the recognition element or a non-reactive recognition element, such as insensitive microparticles, thus the ratio of the readings of the sensitive portion 6′a2 is approximately 0% agglutination reactions. In this embodiment, the concentration of the recognition element with respect to microparticles with increased sensitivity in all given sensitive areas 6a will equally represent the optimized concentration for a specific analysis optimized in this laboratory.

In a further and most preferred embodiment, the two sensitive regions of the first surface 2a of the lower layer 2 are coated with calibration formulations 18 containing different and predetermined concentrations of the analyte or compounds that cause the same or equivalent non-catalytic affinity reaction with the recognition element.

In the most simplified calibration model, the optical response caused by the affinity reaction between the analyte and the recognition element is linear or can be linearized by means of an appropriate mathematical function, such as f (log (x)) or f (1 / x). After injection of the liquid sample using the sampling section 9, the sample will be distributed into the sample-sensitive areas. Here, the sample dissolves the calibration composition 18, coated on the first surface 2a, and the recognition composition 19, coated on the second surface 4a, initiating an affinity reaction between the analyte 31, calibration compound 33 and the recognition element 32. Due to the different concentrations of the calibration composition in the sensitive sections 6′a2 and 6′a3, which is added to the analyte contained in the sample, the measuring device and correspondingly recording device can correlate the optical readings of all sensitive sections 6a with the concentration of the analyte in the sample liquid. The optionally sample-sensitive portion 6c does not contain a recognition element or non-specific binding reagent, such as microparticles without increasing sensitivity, thus applying sample fluid to the sensitive portion 6c does not trigger agglutination and is used to evaluate the background signals of sample fluid 15 in the analyte test element . The described calibration method is known to those skilled in the art as a "standard addition method" and is used in laboratories for samples with a "complex" sample matrix.

FIG. 9 shows various structures of a sample distribution system that can be implemented by hydrophilic channels, as shown in FIG. 6, or by hydrophobic “guide elements”, as shown in FIG. 5, or by a combination of hydrophilic channels and hydrophobic guide elements . Cell A1 in FIG. 9 shows cases for a simple sample distribution system. Column A of FIG. 9 shows the formal design of sample distribution systems without background adjustment, while column B provides structures for sample distribution systems with background adjustment, column C indicates the highest order polynomial calibration equation achievable with adjacent structures, and column n indicates the desired the number of specified sensitive areas of each surface and, accordingly, the number of required measurements. The letters in each design indicate the position of the background correction (s), the sample (1), and all associated calibration sections (2, 3, 4, 5, 6) with increasing number of calibration compounds. The simplest calibration is represented by a linear equation, where the relationship between the measurement and the concentration of the analyte is strictly proportional. Calibration of the test element is mainly carried out using the method of adding a standard by adding a calibration compound of various calibration sections to the sample and then calculating a linear or monotonous nonlinear calibration equation. 13 provides a more detailed explanation of case I. The calibration model or order (column C) must correspond to the selected analyte and the chemical registration method used, therefore, it is impossible to apply a linear calibration model to a chemical reaction that obeys the fourth-order model and vice versa. However, you can still use a test element designed for five standard additives for linear calibration, a larger number of standards provides even more accurate measurement and statistical reliability with higher significance in terms of correlation coefficient, standard deviation and standard error of the test compared to linear calibration, based on two standards.

Moreover, it is also possible to repeat the measurements of the sample and standard, thus, it is possible to perform two independent linear calibrations for one specific sample of a sample of physiological or aqueous liquid with the embodiments shown in row IV. Similarly, you can use the same test element to determine two analytes. Additionally, the system for several analytes can be implemented within the same set of specified sensitive areas if the selected chemical registration reaction does not cause interference problems, thus, the extraction products and products of one reaction do not participate in other reactions, and the resulting indicator in the form of a dye absorbs light, essentially in a different wavelength range.

In additional and alternative embodiments of the test element, the calibration is of a better quality. Here, the result of the sensitive portion 6a1 is compared with the fixed limits of the positive standard 25c and negative standard 26c provided for additional sensitive areas, for example 6a2 and 6a3 (see details in FIG. 12). This method is convenient for analytes that require only high-quality indications, mainly known for pregnancy test systems.

In the case of a non-linear optical response of the reaction between the analyte and the recognition element, other calibration models can be used if the sample distribution system is adopted in accordance with Fig. 9 for a non-linear nature by introducing a larger number of specified sample-sensitive sections in the sample distribution system of the test element. Those skilled in the art will note that FIG. 9 provides examples for sample distribution systems for illustration purposes. Other geometric solutions are also possible and are used to perform similar or related tasks.

The optical response of the reaction between the analyte and the recognition element 32 contained in the recognition part 19 is measured by a measuring device or a recording (detection) device. The measuring device or recording device provides a strip holder with at least one, but preferably with a plurality of arrangements, containing a light source 20 for emitting a wavelength of 24 through a predetermined sample-sensitive section 6, as shown in FIG. 10. The wavelength of 24 light 20 may be in the range of 300-800 nm, but preferably between 400 and 650 nm and most preferably 635 nm. The light emitted by the light source passes through an optical circuit 21, 22, such as a lens and aperture, a lower layer 2, a liquid medium 15, an upper layer 4 of a sensitive portion and is detected on the opposite side of the test element by a recording device 23. Preferably, the light recording device comprises an optical circuit 21a and 22a, such as an aperture, a lens, and / or an optical filter, and at least one of the following: a photodiode, a phototransistor, a photocell, a photomultiplier, or a group of charge-coupled devices. More preferably, the light recording device comprises a photodiode. The light source may be a light emitting diode or a laser diode with an appropriate wavelength.

In the case of an agglutination reaction controlled directly by light scattering and without the help of microparticles, it is most convenient to position the light source and the recording device not opposite each other, but at an angle of approximately 90 degrees to achieve maximum sensitivity. The preferred angle between the light source and the recording device is between 80 and 120 degrees, but the angle of about 109 degrees is most preferred, and the test element is located in the optical recording circuit so that the angle between the lower layer and the light source and the angle between the upper layer and the recording the device is approximately 54 degrees to reduce background noise due to internal reflection on various surfaces of the sensitive area (or more mainly of the test element), as shown in and Fig.11. Thus, the recording device only sees the light wave 24 deflected by scattering on agglutinated particles, and does not see the light wave 24a emitted by the light source. However, those skilled in the art will note that the actual angle must be optimized for the particular application, the required sensitivity, and the requirements for the measuring device relative to the recording device. In addition, the circuit shown in FIG. 11 can be used to detect fluorescence if the light source 20 and the photodetector 23 are configured with additional filters that discriminate between the excitation and emission wavelengths.

In addition, the incidence of light waves in each sample-sensitive region 6 when an affinity reaction occurs causes the scattering of light waves. Since the concentration of the analyte in each sensitive region is different due to the addition of the analyte or the addition of a compound equivalent to the analyte as the calibration compound 33, the optical density of each resulting light beam is also different. If there are at least three sensitive sites located within the sample distribution system, thus at least two sensitive sites contain different levels of the calibration compound, you can calculate the unknown concentration of the analyte 31 in the liquid medium of the sample 15 at a given sensitive area without a calibration connection (6′a1), since the ratio between agglutination and the concentration of the analyte follows a linear or monotonically increasing, respectively, decrease cabbage, models. Potential sample distribution systems corresponding to calibration models based on a polynomial relationship are shown in FIG. 9 as described above.

In the case when the agglutination reaction does not occur, i.e. there is no reaction between the analyte 31 and the recognition element 32 in the sensitive area, the light is scattered, which leads to a pseudo-high optical density (absorption). Conversely, in the case of ~ 100% agglutination, in accordance with the strong reaction between the analyte, the calibration compound, and the recognition element on the first surface of the lower layer and the second surface of the upper layer, the light is scattered minimally, which leads to a low optical density.

On Fig shows examples of absorption graphs depending on the concentration with high-quality registration mode. Here, the specified sensitive sections 6a2 and 6a3 are configured to provide information on the dynamic range of the agglutination reaction, said section 6a2 provides a comparison with ~ 0% agglutination representing the negative standard 26c, respectively, section 6a3 provides a comparison with ~ 100% agglutination representing the positive standard 25c, such thus, the readings of these sensitive areas can be used to confirm the measurement performed on the sensitive area 6a1.

On Fig shows examples of graphs and estimates of absorption depending on the concentration of the agglutination reaction between the analyte and the recognition element in a liquid medium, suitable for quantitative analysis. The concentration of the recognition element, which may be, for example, a predetermined amount of an antibody or an antibody immobilized on microparticles, in sensitive areas 6a1 through 6a3, optimized to achieve the appropriate sensitivity and dynamic range for the analyte of interest. In addition, light scattering or optical absorption of a sample located in the sensitive portion 6c is measured in the fourth and optionally sensitive portion. The readings of this sensitive area provide a quantitative estimate for the background of the sample, providing complete information on the non-specific part of the reaction caused by the materials of the test element, such as films, inactive coating compounds, non-specific proteins or microparticles without increasing their sensitivity or the sample matrix itself, which is especially needed for quantitative sample evaluation.

As mentioned above, coating with a calibration composition 18 containing a calibration compound 33 and a recognition composition 19 containing a recognition element 32, respectively, a recognition element immobilized on respective microparticles 35, can be performed by several known methods, such as microcontact printing, ink dispensing printing or precipitation methods or other capillary contact precipitation methods. The concentration of active components in coatings or dyes, such as a calibration compound and a recognition element within the test element, can be optimized by laboratory methods to determine the acceptable dynamic range and sensitivity of the test system for a particular analyte of interest.

If the dynamic range of a particular test is evaluated in the laboratory, certain characteristics can be programmed in the measuring device or recording device as a confidence window 29, as shown in Fig. 14, to confirm the results after the measurements and, in addition, to evaluate the statistical parameters of the performed calibration calculation 30, such as standard deviation, standard error, and regression coefficient.

Fluorescence based registration methods

The characteristics of the methods described above for analysis of agglutination depend on the speed of analysis and the structure of the measuring device, and these parameters are interrelated. On a molecular scale, the microparticles are huge and approximately larger than the IgG antibody (immunoglobulin G), from 1,000 to 10,000 times, which is a common example for a recognition element. In the case of recognition element (s) 32 immobilized on microparticles 35, diffusion of microparticles is much slower than diffusion of only one recognition element, which affects the analysis time and, therefore, the characteristics of the potential product, but the agglutination reaction can be controlled by a very simple and cost-effective registration device or even with the eye, if the appropriate microparticles are selected, which is due to the fact that the scattering intensity increases by about 1020 if the particle size is increased by a factor 5 th.

On the other hand, the agglutination reaction between the analyte and the non-immobilized recognition element 32 can be successfully controlled at the molecular level by the correct laser-based monitoring devices, as described above. You can even quantify the molecular weight of the recognition element and the resulting agglutinate after reaction with the analyte. However, the cost of the necessary equipment, mainly due to the use of laser technology, interferes with the use of on-site diagnostics or monitoring at home.

An additional embodiment of the invention provides a solution that is midway between the two agglutination methods described. At the molecular level, the agglutination of the recognition element and the analyte creates a network of small protein particles that changes the individual molarity of water and proteins, even though this does not affect the overall mass balance between proteins and water. After the agglutination reaction, some areas in the sample fluid have a higher concentration, and some lower protein concentration compared to the time moments before the agglutination reaction. This change in physical properties can be detected and reported either by fluorescent hardening of dye molecules that have high affinity for proteins, or by fluorescent molecular rotors.

Fluorescent dyes with non-specific protein affinity as a reporter element

The underlying principle of this registration method is the influence of concentration-dependent fluorescence hardening. Above a certain concentration, the fluorescent dye molecules become absorbers of the fluorescence emitted by the molecules of another fluorescent dye, so the total recorded emission of the sample decreases with increasing concentration. This decrease depends on the nature of the fluorescent dye and the direct distance between the two molecules of the fluorescent dye, in other words, the closer the dye molecules and the shorter the distance, the stronger the fluorescent hardening. In a homogeneous solution, the described and well-known effect of fluorescent hardening simply depends on the concentration of the fluorescent dye, since the quenching threshold depends on the fluorescent dye used or a mixture of fluorescent dyes. The hardening effect becomes interesting for recognition analysis only if the dyes are unevenly distributed in the sample liquid and in some way reflect the agglutination of the analyte and the recognition element, resulting in an uneven distribution of the analyte and recognition elements in the sample liquid. Additionally, it is useful to select fluorescent dyes that detect an increased quantum fluorescence yield only after dye absorption on the surface of the proteins to minimize the background signal. Suitable options for this principle of analysis can be found in the class of amphiphilic zwitterionic dyes based on aniline-pyridinium with two homologous series derived from dibutylaniline-pyridinium-butyl sulfonate and dyes based on styrene (also known as hemicyanin dyes), such as RH364, RH160, RH237, RH421, di-4-ANEPBS, BNBIQ, ANNINE-5 and ANNINE-6. Structural formulas of examples of these dyes are shown in Fig.15. However, more well-known colorants such as Nile Blue A, Phloxin B, 1-anilino-8-naphthalene sulfonate (ANS) or their derivatives can also be used for the described method.

The key reaction of this method is the in-situ coating of the recognition element and the analyte inside the sample liquid with the chosen dye, thus, the reaction is limited to the recognition and analyte elements, which are mainly related to the protein family. However, the sequence of events, agglutination reaction and dye uptake are not important, since dye uptake does not interfere with agglutination. During agglutination, the molarity of an individual dye increases inside the agglutinate and causes a hardening effect. The concentration of the dye provided for in the analyte test element should be estimated for each combination of the dye, recognition element and analyte, as well as for other experimental conditions, but mainly depends on the coverage of the dye protein, the constant binding of the dye protein, the compactness of the agglutinate and the application the excitation and emission integral, which is a function of the Stokes frequency shift and the half-width of the passband of the dye fluorescence spectrum.

After applying the liquid sample medium to the sampling section 9, the sample will be distributed by the sample distribution system 6 to the sample sensitive sections 6a. Here, the sample dissolves the calibration composition 19 containing the fluorescent dye and the calibration compound 32 on the surface 2a of the first layer 2, and the recognition composition 18 containing the recognition element 32 on the surface 4a of the second layer 4. After mixing the compounds provided on the first and second surfaces, the reaction begins, the recognition element itself and the analyte, provided that the analyte is a protein, becomes coated with a dye, and the population of molecules of the recognition element, respectively particles of undergoing crosslinking with the analyte. Thus, the degree of fluorescence hardening can be measured by the recording element of the test element. For high-quality registration modes, two sample-sensitive regions are configured as a negative control 6′a2 and a positive control 6′a3. A negative control measurement refers to the fluorescence of the entire contents of the sample proteins plus the added components provided for in the respective sample-sensitive areas without agglutination. To avoid the agglutination reaction, the sensitive portion 6′a2 is covered with a nonspecific recognition element without affinity for the analyte. Therefore, the fluorescence readings of this sensitive region lead to a 100% signal characterizing the upper limit of the dynamic range.

Conversely, the measurement of the positive control, located on the 6′a3 sensitive portion of the sample, refers to the fluorescence of a fully formed and agglutinated accumulation of dye, which provides the maximum possible fluorescence hardening. Such a positive control can be achieved due to the excess of the calibration compound, respectively, of the analyte or the equivalent of the analyte with the calibration composition, coated on the sample sensitive section 6′a3. This configuration not only allows you to report the presence or absence of the analyte, but also allows you to report semi-quantitative information about the concentration of the analyte in relative units compared with the measurements of negative and positive control, such as low, medium and high concentration.

If the ratio between the concentration of the analyte and fluorescent hardening is linear in the desired concentration range of the analyte or can be linearized using the appropriate mathematical formula, quantitative results for the analyte can be obtained if a known amount of the calibration compound is deposited on sensitive sections 6′a2 and 6 ′ a3, thereby the concentration of the calibration compound in the sensitive section 6′a3 is higher than the concentration of the calibration compound neniya 6'a2 on site. In the case when this ratio is non-linear, quantitative information for the analyte can be obtained using test elements of the analyte, providing a higher number of sample-sensitive areas and more than two different concentration levels of the calibration compound (Fig. 9). The processing method used by the processing device to calculate the analyte concentration by the linear method of adding the standard is the same for both examples, in addition to signal pre-processing and scale, as indicated in FIGS. 13A and 13B. Thus, a more detailed study of methods for adding a standard in this example is omitted.

Nonspecific fluorescent molecular rotors as reporter elements for agglutination analysis

The progress of the agglutination reaction leading to agglutinate 34 with associated fluorescent molecular rotors is shown in FIG. from 16 A to 16 C. In this case, the test system uses the effect that the fluorescent molecular rotors 36 increase their fluorescence if the rotation of special groups in the molecular structure is hindered either by an increase in viscosity or due to spatial difficulty. Accordingly, the fluorescence quantum yield of the dye used increases with increasing size of the agglutinate and the level of crosslinking, since both parameters depend on the concentration of the analyte.

Typical fluorescent molecular rotors are based on the main structure of xanthine dyes. Xanthine dyes are obtained by condensation of phthalic anhydride with resorcinol (and derivatives) or m-aminophenol (and derivatives), of which fluorescein is the prototype (all such dyes contain xanthine nuclei). However, not all xanthine dyes are fluorescent molecular rotors. Although fluorescein provides the basic structure for a molecular rotor, it does not provide the desired relationship and relationship between fluorescence and intermolecular rotation of the side groups. This effect does not prevail in fluorescein because of the lateral hydroxyl groups at position 3 ′ and 6 ′ of the molecule, which is affected only by the pH of the sample medium and is not affected by the micro viscosity of the sample liquid.

Effective fluorescent molecular rotors are based on the underlying xanthine structure prevailing in rhodamine dyes. These dyes are obtained by condensation of phthalic anhydride with m-dialkylaminophenols. The hydroxyl groups of (HO-) fluorescein in the rhodamine dyes are replaced by secondary amino groups (R2N-) as effective rotors in the molecular structure. Rhodamine B, sometimes called acid red 52, contains two ethyl groups as R residues in the molecular structure of the rotors and provides the required fluorescence and molecular mobility characteristics used in the test system.

Rhodamine B or sulforodamine B with increased solubility in water is simply an example of a fluorescent molecular rotor for the described application. Dyes based on Auramine O, crystalline violet, pN, N-dimethylaminobenzonitrile, pN, N-dimethylaminobenzylidene malalononitrile, yulolidinbenzylidene malononitrile, Rhodamine 19, Rhodamine 6G, oxazine 1, oxazine 4, oxazine-based dyestuff, rosamine carbone, pyro amine pyrene thiazines are further examples of effective fluorescent molecular rotors. Typical molecular structures are shown in FIG.

To achieve the desired dynamic range and sensitivity with the test system, respectively, optimal tracking of the reaction between the analyte of interest and the corresponding recognition element, the most suitable fluorescent molecular rotor must be selected or even modified with respect to the size of the rotors. However, the modification of fluorescent molecular rotors is included in the scope of the present invention.

A specific fluorescence reporter element using total molecular rotation for competitive and non-competitive recognition analyzes

In an additional embodiment of the present invention, the fluorescent dye is involved in the affinity reaction between the analyte and the recognition element, since the specific dye residue R has an affinity for the recognition element. In accordance with this method, all functional groups, some of the functional groups or part of the functional groups of the dye molecules are replaced by a specific residue R representing the structure of the reagent involved in the reaction, for example, the analyte, peptide, hormone, drug or nucleic acid, such as an oligonucleotide or a short aptamer. The three examples below show possible modifications of fluorescent dyes fluorescein. Rhodamine 6G and Oxazine 4:

In contrast to the fluorescence molecular rotor example above, where a change in intramolecular flexibility changes their quantum fluorescence yield, the currently introduced registration scheme for the test element focuses on the general rotation of the fluorescent dye molecules with a specific affinity for the recognition element. For specialists, it is obvious that the general rotation of the molecules in a given liquid sample medium depends on the hydrodynamic diameter of the molecule, which is associated with the molecular weight of the fluorescent dye. The general rotation of the fluorescent dye molecules can be directly controlled by the polarization of fluorescence, first discovered by Perrin in 1926.

The fluorescence polarization is mainly inversely proportional to the rotation speed of the molecule, so the faster the dye rotates, the lower the observed fluorescence polarization signal, and therefore it is proportional to the molecular weight, corresponding to the apparent hydrodynamic size of the molecule. In other words, the larger the molecule, the stronger the fluorescence polarization signal.

For registration of fluorescence polarization of the linear polarized light used to excite the desired fluorescent dye, and the resulting fluorescence emission is measured after the second polarizing filter, whose polarization plane is oriented either parallel (I _||), or perpendicular (I _⊥) excitation device plane. When such linear polarized light is absorbed by the fluorophore, it can become depolarized, provided that the molecule rotates in the solution at a significant angle over the period of its fluorescence period.

For example, fluorescein, a fluorescent dye with a molecular mass of ~ 332 D, attached to ligands with a molecular mass below 5000 D, returns only a weak fluorescence polarization signal, which is due to the significantly depolarization inherent in the rapid rotation of molecules - more than 4 ns of fluorophore fluorescence time. This means that during the second measurement with filters oriented perpendicular to each other, a significant emission intensity passes through the recording device, so I _{⊥ is} significant and similar in magnitude to I _|| . This results in a relatively low polarization signal.

In the case where the fluorescent dye binds a large recognition element, the degree of rotation of the molecules during the fluorescence is greatly reduced, and the signal depolarization is negligible. Therefore, a low emission intensity is measured with polarizing filters oriented perpendicular to each other, while I _{⊥ is} small, which creates relatively high resulting polarization signals.

To observe fluorescence polarization, the measuring device must be configured with two polarizing filters, one in the optical circuit 21, 22 of the light source 20, and the other in the optical circuit 21a, 22a of the optical recording device 23. The measuring device must measure the fluorescence intensity twice. For one measurement, polarizing filters are oriented parallel to each other (I _|| ); the second measurement is performed with filters oriented perpendicular to each other (I _⊥ ). The devices of the prior art make it possible to select different polarization planes with polarizing filters mounted in a wheel or other mechanical locks, allowing changing rotation or physical displacement. The mechanical selection of filters provides high versatility and some advantages for laboratory equipment. However, this is not suitable for small and portable devices or even handheld devices.

A non-mechanical solution to this problem is to select a parallel or perpendicular plane of polarization by means of a liquid crystal filter or a liquid crystal device for selecting a plane of polarization. A liquid crystal filter or a liquid crystal polarization plane selection device can be considered as a “sandwich” type design of three load-bearing layers parallel to each other, between which are two layers of liquid crystals that can be oriented in the same plane of polarization, applying potential to two adjacent load-bearing layers, covering liquid crystals. The potential applied to two adjacent bearing layers can be controlled by the processing unit of the measuring device. Those skilled in the art are familiar with the relationship of a liquid crystal device for selecting a plane of polarization with a modified liquid crystal display unit.

The processing unit of the measuring device currently allows calculating the fluorescence polarization P from the two measured fluorescence intensities of both polarization planes according to the following formula:

It is obvious from the formula that the polarization P is a dimensionless quantity and, therefore, does not depend on the concentration of the used fluorescent dye, the intensity of the light source and, most importantly, on the attenuation of the fluorescence intensity due to the properties of the sample itself. This registration principle is equally valid for competitive and non-competitive recognition analysis, as described below.

Competitive assays reveal an inversely proportional relationship between the amount of analyte and the fluorescence polarization, thus, a low concentration of the analyte gives a strong polarization of fluorescence, and a high concentration of the analyte gives a low polarization of fluorescence (as shown in figv). Additionally, the recognition element is considered an active binding element or a host molecule, such as an antibody, and the analyte is considered a passive binding element or a guest molecule.

Conversely, a high concentration of the analyte will necessarily give a high fluorescence yield when using the above-described agglutination method with non-specific fluorescent molecular rotors, when the fluorescent molecular rotor is not directly involved, but is forced and impedes rotation by the recognition reaction (as shown in Fig. 12A).

A flow diagram of the reaction between the analyte 31 and the recognition element and the specific reporter element 37 is shown in FIG. After the introduction of sample A) and dissolution of the recognition composition and the calibration composition containing a specific fluorescent dye as a reporter element B), the recognition reaction C) occurs. Now, the analyte 31 contained in the sample medium 15 competes with the specific residues of the fluorescent dye 37 for the existing recognition elements provided on the second surface 4a of the sample-sensitive region, forming a complex grid 38 between all reaction elements. If the concentration of the analyte molecules is high, almost all of the binding sites of the provided recognition element 32 will be occupied, which leads to an unlimited freely rotating specific fluorescent dye 37 with low polarization of fluorescence. Conversely, if the concentration of the analyte molecules in the sample medium is low, the binding sites of the recognition element 32 are mainly occupied by specific residues of the fluorescent dye 37, thus leading to limited dye molecules and high fluorescence polarization. This basic correlation of the concept of competitive analysis is shown in FIG.

However, specialists in this field may consider an additional configuration in which the analyte itself acts as an active binding element, respectively, as a host molecule. This happens when the analyte of interest is an antibody, i.e. if the patient is tested for his response to vaccination. In this case, a specific fluorescent dye reacts directly with the analyte without the participation of another reaction element, so the reaction is non-competitive, and the fluorescence polarization will be proportional to the concentration of the analyte in the dynamic range of the reaction. This case is shown in figa.

The basic design of the test element is based on the above description. In addition, the test element can be configured to obtain qualitative results or quantitative results. Qualitative results provide information on the presence or absence of the analyte, or information on whether the analyte is present in low or high concentrations, which can be achieved by measuring with positive and negative controls to determine the dynamic range of the test system. Accordingly, the sensitive portion 6′a2 is configured for negative control when the fluorescent dye is not limited by the affinity reaction, so the recording device can evaluate the background fluorescence (in the case of the competitive method, this will be a positive control). This condition can be achieved by using a non-specific recognition element in the sensitive area 6a2.

The sensitive section 6a3 is configured for a positive control when the fluorescent dye becomes completely bounded by either fully formed agglutinate, or if the fluorescent dye is completely captured by the recognition element, so the test system recording device can detect maximum fluorescence (in the case of a competitive method, this will be a negative control) . Thus, the calibration composition contains enough analyte or calibration compound to trigger a fully completed affinity reaction.

Quantitative results are achieved with the test element of the analyte if at least two sample-sensitive sections are configured with at least two different concentrations of the calibration compound in the calibration composition. The minimum number of two different levels of calibration compounds should be sufficient to evaluate analytes with linear response characteristics. If this characteristic is more complex, the number of sample-sensitive sections, respectively, the number of different calibration compositions should be increased to satisfy the requirements of suitable calibration models, such as the equation of higher order polynomials.

When these requirements are met for a competitive linear model, a calibration composition containing a specific fluorescent dye and a calibration compound, such as an analyte or a molecule equivalent to the analyte, is coated on the sensitive areas 6′a2 and 6′a3 of surface 2a of the first layer 2. Calibration composition, coated on a sample-sensitive region 6′a1, contains only a specific fluorescent dye as a reporter element, but does not contain an additional libration compound. Sensitive sections 6a1 through 6a3 on the surface 4a of the second layer 4 are coated with a recognition composition containing a recognition element in the desired concentration.

In the case of a non-competitive method, a specific fluorescent dye becomes a recognition element and is coated on a sensitive area on the opposite side of the calibration compound. To clarify the configuration for linear competitive and linear non-competitive test systems, see Table 1.

Table 1 Compositions of various coatings required for various sample sensitive sites in competitive and non-competitive analysis Position in the sample distribution system Competitive analysis Noncompetitive analysis 6a1 (1st surface) Level 0
calibration composition: only specific fluorescent dye Level 0
calibration composition = control composition 6′a1 (2nd surface) Recognition composition: actively binding component, i.e., an antibody as a recognition element Recognition Composition: Specific Fluorescent Dye Position in the sample distribution system Competitive analysis Noncompetitive analysis 6a2 (1st surface) Level 1 calibration composition: analyte or equivalent with a first concentration as a calibration compound, specific fluorescent dye Level 1 calibration composition: analyte or equivalent with first concentration as calibration compound 6′a2 (2nd surface) Recognition Composition: Actively binding component, i.e. antibody as recognition element Recognition Composition: Specific Fluorescent Dye 6a3 (1st surface) Level 2 Calibration composition: analyte or equivalent with a second concentration as a calibration compound, specific fluorescent dye Level 2
Calibration composition: analyte or equivalent with a second concentration as a calibration compound 6′a3 (2nd surface) Recognition Composition: Actively binding component, i.e. antibody as recognition element Recognition Composition: Specific Fluorescent Dye 6s (1st surface) The control composition does not contain a fluorescent dye or recognition element for the analyte The control composition does not contain a fluorescent dye or calibration compound 6′s (2nd surface) The control composition does not contain a fluorescent dye or recognition element for the analyte The control composition does not contain a fluorescent dye or calibration compound

Apoenzyme Recognition Analysis Tool (AARA) as a reporter element

The apoenzyme recognition analysis method (AARA) can be used to detect the presence of an analyte in a liquid medium without physically separating free and bound species by washing, which is a common drawback of immunological assays, such as enzyme linked immunosorbent assay (ELISA), which impedes the introduction of this concept into universal test system. However, the AARA method may be limited to small analytes such as hormones, steroids, peptides (e.g., B-type natriuretic peptide (BNP)), cardiac markers such as troponin I (TnI), troponin T (TnT) and troponin C (TnC) , haptens, organic molecules such as pesticides, drugs (especially if used improperly: amphetamines, barbiturates, benzodiazepines, cocaine, methamphetamines, opiates, phencyclidine, TNS) or explosives. Some of the known and used tumor markers in the diagnostic field, for example, a carcinoembryonic antigen (CEA) with approximately 180,000 daltons, may be too large for this concept of analysis.

19 shows the AARA principle mechanism used in the test system of the present invention with a recognition element 32 (e.g., an antibody), where the analyte 39, labeled with coenzyme, is capable of reacting with the corresponding apoenzyme 40 to form the enzymatic active holoenzyme 41. In the quantification of the analyte by AARA, the fact that the concentration of the analyte 31 is proportional to the restored holoenzyme concentration, respectively modulated active STI enzyme for holoenzyme obtained by reduction of labeled analytes coenzyme and apoenzyme.

In a specific example, the coenzyme may be a flavin adenine dinucleotide (FAD) coupled to a derivative of the analyte of interest, for example BNP, since the glucose oxidase apoenzyme is used as part of a reporter element. In the absence of a free analyte, the labeled coenzyme reacts with the recognition element, in this case BNP, with a specific antibody, thus inhibiting the restoration of the holoenzyme. The more free analyte (for example, BNP) is contained in the liquid medium of the sample, the more proportionally the recognition element becomes captured by the analyte, and the proportionally more labeled coenzyme becomes available to restore the holoenzyme. For the described mechanism, it is important that labeled coenzyme 39 becomes a spatially protected recognition element 32, preventing recovery with apoenzyme 40. As is well known to those skilled in the art, activated GOD begins to oxidize glucose to gluconolactone and hydrogen peroxide, which can be used for subsequent and corresponding chromogenic reactions. The test element is designed with at least two, but preferably three, sample sensitive sites for performing AARA. The design corresponds to the above description, providing for the lower layer 2 and the upper layer 4 at a predetermined distance opposite each other with a sample distribution system 6 on surfaces 2a and 4a. With some polymer films, it may be preferable to provide a sample distribution system 6 not only with a hydrophilic coating 17, but also with hydrophobic guide elements 16 to achieve an optimal sample distribution.

The main difference between the above embodiments for agglutination is the coating of the active components and their combinations on the first and second surfaces. The AARA method requires more than two, in this case four active components plus chromogenic reporter elements containing enzymes such as horseradish peroxidase and enzymatic dye substrates. It is important to combine only inactive combinations of components on the same surface of sensitive areas. These inactive combinations are a mixture of two or more chemical compounds that do not react with each other, such as two enzymatic substrates, since the active combination is a mixture of two reactive compounds, such as an enzyme and the corresponding enzymatic substrate or recognition element (t ie, antibody) and the corresponding analyte (e.g., antigen). These inactive combinations, provided on the same surface as a composition or printing ink, dissolve upon contact with the liquid medium of the sample and, therefore, form an active combination with compounds of the opposite surface after mixing, which triggers a chemical or physical reaction.

According to this method, each sample-sensitive portion of the first surface is coated with calibration formulations that contain the analyte or an equivalent molecule labeled with the analyte coenzyme, plus the compounds required for photometric evaluation, such as the chromogen and enzymatic substrate required by the holoenzyme. Depending on the principle of calibration, the concentration of the calibration compound in the calibration composition changes by n-1 or n-2 the number of sample-sensitive sections, so at least one sample-sensitive section does not carry a calibration compound. Sites sensitive to the sample on the second surface are coated with a recognition composition containing a recognition element for recognizing the analyte of interest, apoenzyme, plus additional enzymes, if required for the chromatogenic reaction.

AARA Standard Addition Method

As mentioned above, the role of determining whether an analyte exists in a liquid medium is important to doctors. It is also important to determine the concentration of the analyte in a liquid medium. In one embodiment of the present invention, a test element is configured to quantify an analyte of interest based on a linear standard addition method. For this example, we consider the linear ratio of the analyte in the desired monitoring range of the analyte.

Similar to the AARA method described above, the test element has four sensitive sections on the first surface and the second surface, combined essentially congruently to create a sample distribution system. Three sample-sensitive areas (6′a1, 6′a2, 6′a3) are covered by the calibration composition 18, since the calibration composition, coated on the sample-sensitive sections 6′a2, 6′a3, contains two different preset and known concentrations of the calibration Compounds 33. In contrast, the sample-sensitive portion 6′a1 is coated with a calibration composition that does not contain a calibration compound or analyte. The sample-sensitive section 6′s is not covered by the calibration compound and is used to evaluate the background of the sample. Sites sensitive to the sample (6a1, 6a2, 6a3) on the second surface 4a are coated with a recognition composition 19 provided for a given concentration of the recognition element 32, apoenzyme and, if necessary, additional enzymes. The sample-sensitive portion 6c, similarly to the oppositely located portion 6′c, ensures the absence of a recognition composition or composition without active components, respectively, of a recognition element. Table 2 provides an overview of the above configuration.

table 2 Functional components for calibration and recognition compositions required for analysis of recognition using apoenzyme Position in the sample distribution system 1st surface
Functional compounds of calibration compounds 2nd surface
Recognition Functional Compounds 6′a1 / 6a1 coenzyme-labeled analyte, enzymatic substrates, chromogens recognition element (i.e. antibody specific for the analyte), apoenzyme, additional enzymes, if required for optical and photometric registration 6′a2 / 6a2 Specified calibration compounds C = 1 (i.e., analyte or equivalent), coenzyme-labeled analyte, enzymatic substrates, chromogens recognition element (i.e. antibody specific for the analyte), apoenzyme, additional enzymes, if required for optical and photometric registration Position in the sample distribution system 1st surface
Functional compounds of calibration compounds 2nd surface
Recognition Functional Compounds 6′a3 / 6a3 Specified calibration compounds C = 2 (i.e. analyte or equivalent), coenzyme labeled analyte, enzymatic supports, chromatogens recognition element (i.e. antibody specific for the analyte), apoenzyme, additional enzymes, if required for optical and photometric registration 6′s / 6s uncoated or inert coating without active ingredients uncoated or inert coating without active ingredients

After applying the liquid medium to the sample distribution system, which ensures the re-dissolution of the calibration composition and recognition composition and subsequent reactions between them, the light source 20 and the registration circuit of the measuring device 23 measure the first optical absorption 25a of the liquid medium located in the sample-sensitive section 6′a2 with the first level of the calibration connection, as shown in figa. The readings of this sensitive section 6a2 are a signal proportional to the combined concentration of the calibration compound in section 6′a2 and the concentration of the analyte in the liquid medium 15. In parallel, the second optical absorption 26a is measured for the liquid medium 15 of the sample located on the sensitive section 6a3 with the second level of the calibration compound in section 6′3, representing a signal proportional to the combined concentration of the calibration compound and the concentration of the analyte. In addition, the third optical absorption 27a is determined by the sensitive portion 6a1 not containing the calibration compound in the calibration composition deposited on the sensitive portion 6′a1. Thus, the liquid medium 15 of the sample contains only an unknown concentration of the analyte 28. According to the theory of the method of linear addition of the standard, the processing system of the test system can calculate the measured absorption 25a, 26a, 27a and known concentrations of the calibration compound in the sample-sensitive sections 6′a2 , 6′3, unknown concentration of analyte, coefficients for the calibration equation y = c0 + c1x, correlation coefficient and standard deviation for performing analysis by linear p gressionnogo analysis. The specific concentration of the various components contained in the recognition composition and the various calibration compositions according to Table 2 should be optimized through a standard laboratory experiment to achieve optimal test system performance. In addition, if the relationship between optical absorption and analyte concentration exhibits a non-linear characteristic, the method of adding a standard can be modified and applied to such non-linear behavior, providing more sample-sensitive areas, as shown in Fig. 9.

A method of manufacturing a test element

The test element of the present invention, which is preferably made in the form of strips, can be easily manufactured by printing, punching and laminating methods customary to those skilled in the art. The design of the test element can be made simple and cost-effective way, which is preferably, but not necessarily, continuous.

At the first stage of manufacturing, the structure of the sample distribution system is formed by creating a site with high and low surface energy on the substrate. In the first embodiment, high surface energy regions forming sample channels and sensitive regions on the first and second surfaces are created by applying a hydrophilic composition to the hydrophobic surface of the substrate. As described in detail above, it is also possible to create areas with high and low surface energy by applying a structure of hydrophobic "guide elements" to the hydrophilic surface. In a preferred case, the substrate has intermediate hydrophobic properties of a commercially available transparent polymer film, since areas of low and high surface energy of the sample distribution system and sample-sensitive areas are created by printing hydrophilic channels under or surrounded by a hydrophobic structure of hydrophobic guide elements.

The substrate can be made of a material such as glass, polyvinyl acetate, polymethyl methacrylate, polydimethylsiloxane, polystyrenes, polyesters and polyester resins containing fluorene rings, polycarbonates and grafted polycarbonate-polystyrene copolymers, terminal modified polycarbonates, polyolefins, copolyolefins and cycloolene copolymers of olefin-maleinimide.

The application of the hydrophilic structure to the hydrophobic substrate and (or) the application of the hydrophobic "guide elements" to the hydrophilic substrate or any combination thereof can be carried out by contact printing and non-contact printing, spraying, immersion or plasma deposition, in particular, flexography, lithography, intaglio printing, methods of transferring onto a solid dye base or inkjet printing.

However, the preferred manufacturing method is a flexography method, which allows high-resolution printing on rotary presses and provides high productivity. This method is widely used for printing on polymer films and is widely used in the packaging industry. The optical recording method shown in FIGS. 10 and 11 requires a transparent and clean low viscosity ink for a hydrophilic structure. Low viscosity paints are preferred for obtaining a thin and even coating of a thickness of about 2-4 microns. The optical window of the spectrum of the paint should be in the wavelength range suitable for optical registration of chemical reactions. The requirements for hydrophobic paint, in addition to hydrophobic properties, are less stringent, and it can also be used to decorate the test element with the right color, so opaque paint is preferred for this step. The use of a four-color flexographic printing machine is widespread and its use, in essence, will not create problems. The same applies to the lithographic device.

The most suitable for the manufacture of a test element of the solvent-based paint, which are widely represented by various manufacturers. Additionally, all available printing inks can be selected with additional additives and pigments to optimize the required parameters. Many of these paints are based on mixtures of nitrocellulose ethanol or polyvinyl butyral ethanol and can be obtained, for example, from Sun Chemicals Inc. (35 Waterview Boulevard, Parsippany, New York, USA) or Flint Ink Inc. (4600 Arrowhead Drive, Ann Arbor, Michigan, USA).

Although solvent-based paints or cured by ultraviolet radiation are suitable for the manufacture of a test element, paints fixed by an electron beam (EF) have some preferred properties. These paints provide the highest resistance to mechanical and chemical factors and contain 100% polymers, optionally with pigments, but without volatile organic solvents and photoinhibitors, which have been proven to affect stability when using sensors. These positive achievements in improving the performance were obtained due to the ability of electrons to create crosslinked polymer films and penetrate deep into the surface.

Printing inks, which are fixed in the flow of electrons, allow you to use the ability of acrylic monomers and oligomers to polymerize. The use of acrylic compounds for the preparation of printing inks is currently becoming increasingly relevant (see J.T. Kunjappu. "The Emergence of Polyacrylatcs in Ink Chemistry," Ink World, February 1999, p. 40). The simplest acrylic compound, namely acrylic acid, has the following formula (I):

The double bond in the acrylic fragment when interacting with electrons (initiation) opens and forms a free radical, as a result of the interaction of which with other chain-forming (chain development) monomers high molecular weight polymers are formed. As already noted above, radiation polymerization does not require any external initiator, since high energy radiation itself generates free radicals, so that no initiator residues remain in the coating.

Acrylic monomers that are fixed in an electron stream include a large number of acrylic monomers, ranging from simple acrylates such as 2-phenoxyethyl acrylate and isooctyl acrylate to prepolymers such as epoxy acrylate bisphenol A and acrylates / polyesters (R. Golden. J. Coatings Technol., 69, 1997, p. 83). A similar (electron-beam) method of fixing printing inks allows you to create "functional printing inks" with certain chemical and physical properties without the use of solvent and the fixing systems necessary when working with other printing inks, which significantly complicate the entire technological process of manufacturing the test elements proposed in the invention .

Acrylic monomers that are fixed in an electron stream include a large number of acrylic monomers, ranging from simple acrylates such as 2-phenoxyethyl acrylate and isooctyl acrylate to prepolymers such as epoxy acrylate bisphenol A and acrylates / polyesters (R. Golden. J. Coatings Technol., 69, 1997, p. 83). A similar (electron-beam) method of fixing printing inks allows you to create "functional printing inks" with certain chemical and physical properties without the use of solvent and the fixing systems necessary when working with other printing inks, which significantly complicate the entire technological process of manufacturing the test elements proposed in the invention .

Inks with hydrophilic properties can be obtained by selecting polymers soluble in ethanol and water and mixtures of these polymers. Polymers and derivatives of polymers, copolymers and compounds based on alginate, cellulose and cellulose ester, hydroxyethyl cellulose, resins, acrylic acid, polyvinyl alcohol, polyethylene glycol, polyethylene oxide, vinylpyrrolidone, polystyrene sulfonate, poly (methyl vinyl ester), malein can be used copolymers of vinylpyrrolidone / trimethylammonium and derivatives of alkyl phosphocholine. Additional optimization is possible due to the use of organo-modified silicone acrylate additives, which are crosslinkable varieties of organo-modified polysiloxanes, and fluorinated surfactants. A common suitable coating typically provides a contact angle with water of less than 35 ° and a surface energy of more than 50 mN / m.

The second stage of manufacture includes the application of recognition compositions containing a recognition element and additional reagents to obtain printing and (or) dosed inks that form a uniform layer within the areas sensitive to the sample.

On all relevant sample-sensitive sections of the opposite surface, calibration formulations were deposited containing the desired amount of calibration compound required for quantitative or qualitative recording modes. Depending on the registration mechanism, for example, agglutination, fluorescence agglutination or AARA reporter, additional reporter elements can be added either to the recognition composition or to the calibration composition, if necessary, a detailed description is given above and in Table 1.

Since the concentration level corresponding to the total number of recognition element deposited on the given sample sensitive areas 6a1 to 6a3 is responsible for the sensitivity and dynamic range of the various test elements described for the analyte, as well as the concentration level and accuracy of the applied calibration compound are responsible for the accuracy of the results test, for this application it is of paramount importance to provide test elements with an accurate dosage of the above elements, compounds and nents. Accurate dosing can be achieved, for example, by using the microdosing system offered by Vermes Technik GmbH & Co. KG, Palnkamer Str. 18-20, D-83624 Otterfing, Germany. This system also allows dosing of nano- and microparticles, as is necessary for some agglutination reactions. The composition of the coating should be readily soluble in the liquid medium of the sample in order to ensure rapid recovery without residue after the introduction of the liquid sample.

The next step involves the lamination procedure, in which the lower and upper layers, which are the first and second surfaces of the sample distribution system, are layered on the central layer, thereby determining the distance between the first and second surfaces of the lower and upper layers. The central layer provides a cutout to create a cavity for the sample distribution system in areas where the sample distribution system is formed on the first and second surfaces of the lower and upper layers. The structure with high and low surface energy formed on the first and second surfaces of the lower and upper layers should be combined almost congruently to ensure the formation of a functional system for the distribution of samples between the first and second surfaces.

Accurate hu-installation (fixation) of the lower and upper layers becomes critical for the functioning of the element, if this is not achieved, the distribution system of the sample will not function correctly and (or) have a large deviation with respect to the specified volume of the sample. Permissible deviation limits should be +/- 5% of the width of the hydrophilic channels to achieve reliable performance.

On Fig shows a top view (left) and a cross-sectional view (right) of the test element and the impact of installation quality. In case 20a, the sample distribution system is assembled correctly with the reliable combination of the hydrophilic channels of the first 2a and second 4a surfaces. The result of an incorrectly combined test element is shown in FIG. Although the spacer between the lower (2) and upper layer (4) is the same in cases 20a and 20b, the sample volume is erroneously increased in case (b), since the sample liquid partially covers the hydrophobic guide elements of the sample distribution system. This is caused by the sample fluid inside the test element, which tends to have a minimum surface area bordering the atmosphere in order to achieve the most preferred energy state and, therefore, overcome the influence of hydrophobic sites.

In an alternative embodiment, as shown in FIG. 20c, the sample distribution system of the upper layer 4 is planned to be approximately 10% smaller than the sample distribution system of the lower layer 2, thus the total sample volume of the test element is determined by the protrusions of the sample distribution system of the lower layer providing large allowable limits for registration during manufacture without compromising the accuracy of the required sample volume. For specialists in this field it is obvious that the lower and upper layers are interchangeable in the described embodiment, without prejudice to the essence of the invention.

The application of the central layer, which can be a double-sided adhesive tape, preferably 80 microns thick, or alternatively, in the alternative, adhesive adhesive applied with the same thickness, is simplified due to the relatively large cutout in the material compared to the size of the hydrophilic channels. Correct installation is especially important on continuous production lines where the substrate comes with several measuring devices up to dozens of measuring devices per minute. The protrusions of the substrate and the tension of the web make it difficult to install in the x-direction (the direction of movement of the web) compared with the y-direction perpendicular to the movement of the web.

A method of manufacturing flexible polymer films, providing accurate placement of the structures of the first and second surfaces, shown in Fig.21, which indicates the steps of continuous production of the canvas. At the first stage of the production of FIG. 21a, the structures of the distribution system 6 of the samples of the lower and upper layers are printed on a single web substrate 49, which is the material of the test elements of the analyte being created. As shown in FIG. 21, the printed structures of the sample distribution system 6 are located on the substrate web 49 so that the two sample distribution systems are opposite each other to the left and right of the mirror line. Optionally, a sample distribution system is attached at sites that form sample application sites. Thus, the position of the given sensitive sections 6a, 6′a is fixed relative to each other and does not depend on the protrusions of the material and the tension of the canvas.

The dashed lines 50 indicate the lines of the future section for dividing the test elements into strips, while the dashed lines 51 indicate the specular line of the strip blank and the future fold line of the web substrate.

After printing the flow paths of the test element, the sensitive sections 6a, 6′a of the sample distribution system are covered with a calibration composition and a recognition composition. For example, the sensitive sections 6′a of the lower row of the web substrate 49, which represent the first surface of the test element, are coated with a calibration composition comprising a reporter element and various levels of calibration compound; one of the calibration compositions (for example, located in section 6′a1) does not contain a calibration compound and provides indications of the liquid medium of the sample, while the sensitive sections 6a of the upper row of the substrate 49 of the web, which represents the second surface of the test element, are coated with recognition compositions containing the element recognition, and in the special case of apoenzyme recognition analysis are also additional parts of the reporter system.

Accordingly, an additional layer is superimposed on one of the surfaces, for example, the surface 2a of the lower layer 2, representing the Central layer 52 of the test element, as shown in figv. The center layer 52 may be made of double-sided adhesive tape or adhesive glue that provides breakouts 5 that open the sample distribution system 6 to create cavities for the sample distribution systems in the test elements of the analyte after the final assembly step.

The test element of the present invention is then assembled by folding two sides along the mirror line 51, for example, using a bending apparatus or other suitable equipment, as shown in FIG. 21c, creating a bent and laminated web 53, as shown in FIG. Accordingly, the center layer, the lower and upper layers can be tightly joined by the pressure roller.

Finally, the laminated web 53 is cut out or carved into the product of the desired shape, while line 50 transfers the approximate shape of the final test strip to the web 53 before separation. In the manufacturing method shown in Fig. 21, the upper part of the substrate can be bent to the lower part without the risk of deterioration of fixation in the x-direction of the web and provides an easier way to achieve the correct installation of the first and second surfaces forming the sample distribution system, compared to the method with one layer.

The present invention provides a system for determining the concentration of clinically available and biochemical analytes that are available in laboratories supporting immunological or similar affinity assay analytical methods, such as radio, diffusion or immunological side-stream analyzes, respectively, enzyme-linked immunosorbent assays. Typically, the analyte is recorded in a liquid sample medium, such as blood, serum, plasma, saliva, urine, interstitial and / or intracellular fluid. The test element is equipped with an integrated calibration system and a quality control system suitable for the format of the test strip with dry reagents with a very small sample volume of approximately 0.5 μl. The manufacture of the test element of the present invention does not require many technologically complex operations and allows the manufacture of inexpensive test elements.

Claims (26)

1. A test element for the qualitative and (or) quantitative determination of at least one analyte in a sample of physiological or aqueous liquid, having first and second surfaces (2a and 4a) located at a given distance from each other and both containing two essentially the same structure, forming sections with high and low surface energy, aligned mainly congruently with each other, so that areas with high surface energy form a system (6) of distribution of the sample with at least at least two sensitive areas (6a, 6a), and at least one of the sensitive areas (6a, 6a) of the first and second surfaces (2a, 4a) is provided with at least one non-enzymatic recognition element (32) capable of provide an affinity reaction with said at least one analyte. 2. The test element according to claim 1, in which
n given sensitive sections (6a) of the first surface (2a) are covered with n calibration compounds (18) made from m control compositions and nm compositions containing different levels of the calibration compound (33), where n is an integer greater than 2, m is an integer equal to or greater than 1, and n> m, and
n predetermined sensitive areas (6′a) of the second surface (4a) are coated with a composition containing a non-enzymatic recognition element (32). 3. The test element according to claim 2, having an additional sensitive section (6 s) with non-specific materials that do not contain either an analyte recognition element (32) or a calibration compound (33), providing measurement of background signals. 4. The test element according to claim 2, in which the calibration compound (33) is identical or essentially equivalent to the analyte. 5. The test element of at least claim 1, wherein the specific recognition element (s) (32) is immobilized (s) on the microparticles. 6. The test element according to one of the preceding paragraphs, in which the recognition composition and (or) the calibration composition contains (at) the reporter element (s), which contribute to the optical registration / determination of the analyte. 7. The test element according to claim 6, in which the reporter element is a fluorescent dye. 8. The test element according to claim 7, in which the fluorescent dye has an affinity for the recognition element. 9. The test element according to claim 7, in which the fluorescent dye has an affinity for the analyte. 10. The test element according to claim 6, in which the reporter element is a fluorescent molecular rotor. 11. The test element according to claim 6, in which the reporter elements contain means for analyzing recognition using an apoenzyme. 12. The test element according to claim 11, in which the means for analyzing recognition using an apoenzyme comprises a recognition element, an apoenzyme labeled with a coenzyme to be analyzed, a substrate necessary for the holoenzyme and chromogen. 13. The test element according to one of claims 1 to 5 or 7-12, in which the recognition element (32) is an antibody or antibody fragments to an analyte. 14. The test element according to one of claims 1 to 5 or 7-12, in which the specific recognition element (32) is a nucleic acid. 15. The test element according to one of claims 1 to 5 or 7-12, wherein the specific recognition element (32) is the analyte receptor. 16. A method of manufacturing a test element for analysis of a substance, comprising the following steps:
the formation of areas with high and low surface energy on the lower layer (2) with the first surface (2a), and areas with high surface energy form a hydrophilic system (6) for the distribution of the sample with n given sensitive areas (6′a), where
n is an integer greater than 2,
the formation of the corresponding structure of areas with high and low surface energy on the upper layer (4) with the second surface (4a), coverage of n given sensitive sections (6a) of the first surface (2a) n with calibration compositions (18) made of m control compositions and nm compositions containing different levels of the calibration compound (33), where n is an integer greater than 2, m is an integer equal to or greater than 1, and n> m,
coating n predetermined sensitive areas (6′a) of the second surface (4a) with the recognition composition (19) containing the recognition element (32), and
overlapping layers of the first and second surfaces on opposite sides of the central layer (3) with a cutout (5) providing a cavity for the sample distribution system formed by areas with high surface energy (6, 6 ′) on the first and second surfaces (2a, 4a) of the lower and upper layers (2, 4),
moreover, the recognition element (32) is capable of providing an affinity reaction with the analyte in a sample of physiological or aqueous fluid delivered to sensitive areas (6a, 6′a). 17. The method according to clause 16, in which areas with high surface energy (6, 6 ′) are created by applying a hydrophilic composition to the first and second surfaces (2a, 4a). 18. The method according to clause 16, in which areas with low surface energy are created by applying a hydrophobic composition to the first and second surfaces (2A, 4A). 19. The method according to 17 or 18, in which the hydrophilic and (or) hydrophobic composition (s) are applied to the first and second surfaces by contact and non-contact printing, spraying, immersion or plasma deposition, in particular, flexography, lithography, gravure printing , transfer methods on the basis of solid dye or inkjet printing. 20. The method according to one of paragraphs.16-18, in which the recognition composition and (or) the calibration composition (s) (18, 19) are applied as a coating to the sensitive areas (6a, 6′a) of the first and second surfaces by means of a microcontact printing, micro-spraying or inkjet printing. 21. The method according to one of paragraphs.16-18, in which the lower layer (2) and the upper layer (4) are formed from one flexible substrate and bent along a longitudinal mirror line (45) with the coverage of the Central layer (3) so that hydrophilic structures (6, 6 ′) forming a sample distribution system with predetermined sensitive areas (6′a, 6a) are aligned and fixed essentially congruently. 22. A test system for the qualitative and (or) quantitative determination of the analyte in a sample of physiological or aqueous liquid, containing
a test element according to one of claims 1 to 15, in which
n preset sensitive sections (6a) of the first surface (2a) are covered with n calibration compositions (18) made from m control compositions and nm compositions containing different levels of the calibration compound (33), and n given sensitive sections (6′a) of the second surface (4a) are coated with a composition containing the non-enzymatic recognition element (32), and where n is an integer greater than 2, m is an integer equal to or greater than 1, and n> m,
a recording device for recording changes in the optical properties of a physiological sample located on 2n predetermined sensitive areas and providing n results from 2n predetermined sensitive areas, and
a processing device for calculating all calibration coefficients of the polynomial calibration equation obtained from n measurements, and one regression coefficient to confirm the quality of the calculated calibration coefficients of the calibration equation. 23. The test system according to item 22, containing a device for selecting a plane of polarization. 24. The test system of claim 23, wherein the device for selecting the polarization plane is controlled by the processing device. 25. The test system according to item 23 or 24, in which the device for selecting the plane of polarization is made of at least one liquid crystal unit. 26. A method for the qualitative and (or) quantitative determination of at least one analyte in a sample of physiological or aqueous liquid, including:
applying a physiological sample liquid to a test element according to any one of claims 1 to 15,
connection of the test element to the registration and processing devices, registration and correlation of signals received at various sensitive sites.

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