Fast biosensor with reagent layer

FIELD: chemistry.

SUBSTANCE: detection system for detecting target molecules includes a sensor chip (1), having on its detecting surface (33) an immobilised target molecule or a capturing molecule for target molecules and a soluble reagent layer (5), having a labelled molecule for binding with the target. The group of inventions also relates to a sensor chip (1) and a method of detecting target molecules in a sample using said sensor chip.

EFFECT: high sensitivity and accuracy of analysis while cutting duration of analysis.

19 cl, 8 dwg, 2 ex

The technical field to which the invention relates.

The present invention relates to the field of biosensors. In particular, the present invention relates to methods and systems for registration of the investigated substances used for qualitative or quantitative registration of biological, chemical or biochemical particles, as well as with the means of improving such methods and systems registration.

Prior art

Usually biosensors are devices that allow to qualitatively or quantitatively detect target molecules, also called analytes, such as proteins, viruses, bacteria, cell components, cell membranes, spores, DNA, RNA, etc., in liquids, such as, for example, blood, serum, plasma, saliva, tissue extract, interstitial fluid extract, a cell culture, nutrient extract drinking water.

In almost all cases, the biosensor includes a surface on which there are specific recording elements to capture the analyte. Therefore, the surface of the touch device can be modified by addition of specific molecules capable of binding to target molecules that are in the liquid.

One method of measurement consists of counting the marked molecules, contacting ODA is divided areas of the biosensor. For example, molecules can be tagged with magnetic particles or particles, and these particles or particles can be detected by the magnetic sensor. In another embodiment, the amount of analyte can be determined using fluorescence. In this case, the analyte can carry a fluorescent label, or, in another embodiment, may be an additional incubation with fluorescence labeled secondary recording element.

In most of the biosensors a sensor chip provided with a dry reagent and the surface of the detector is coated with a biologically active surface layer. The reagent may, for example, include tags associated with biologically active agents, such as antibodies to medicine. When the test fluid begins to flow, the dry reagent is dissolved and mixed with liquid. After that the liquid is transported to the sensor surface and wets the surface of the sensor. Labels, as well as the sensor is exposed to molecules of the drug. This affects the binding of registered marks with the surface of the sensor.

Electrochemical biosensors are known, for example, from US 20050016844 A1, in the form of test strips, in which a dry soluble layer deposited near the electrodes or on them. The layer typically includes chemical components for reaction with the analyte or molecule-target, Thu is to create an electrochemical signal, to indicate the presence of analyte in a liquid sample, such as one or more enzymes, coenzymes, cofactors, buffer salts and excipients to enhance the properties or characteristics of the reagents, such as de - and degidratatsiya. The latter is usually used for registration of analytes present in relatively high concentrations in the sample.

The invention

The purpose of the present invention is to obtain good methods and systems for registration of biological, chemical and/or biochemical particles.

This goal is achieved using the following devices and methods.

The present invention relates to a registration system for registering at least one of a target molecule, and detecting system includes at least one of a sensor chip and is designed for receiving at least one label for registration on the basis of the label referred to a sensor chip contains a recording surface with at least one soluble layer containing a reagent, this soluble layer provides the interaction of at least one tag with at least one molecule of a target, thus allowing the signal about registration on the basis of the label.

An advantage of embodiments of the present invention that the fast reg is managed with high sensitivity is achieved through the use of biosensors based on labels. An advantage of certain embodiments of the present invention that can be achieved rapid dissolution of the reagent. Also an advantage of certain embodiments of the present invention that can be achieved by rapid mixing of the reagent with the liquid. Also an advantage of certain embodiments of the present invention in that the reagent is located close to the registration area. In addition, an advantage of certain embodiments of the present invention that a high concentration of mobile reagent can be obtained in the field of registration, which allows you to attain higher levels of binding or nesvyazannie on the surface of the sensor.

An advantage of certain embodiments of the present invention is the ease of processing. As a sensor chip and the recording system can contain all you need to register, no need to provide any liquid, except sample.

An advantage of certain embodiments of the present invention is that testing requires a very small sample volume. An advantage of certain embodiments of the present invention that the recording system may contain a matrix of sensor chips for the so-called touch is ultiplicative, which allows independent analyses in parallel. Various sensors can be used to multianalyte registration, for example, to serve as positive or negative control, calibration purposes and/or for parallel screening. An advantage of certain embodiments of the present invention that the problem of cross-reactivity or cross-contamination is eliminated or reduced because of reagents is not enough time to reach the neighboring sensors due to the limited thickness of the layer of reagent and small duration of the test.

The registration system may include any suitable detector for registering at least one label, the magnetic or optical particularly preferred. The advantage of this system is that samples with a low concentration of analyte, that is, for example, below 1 mmol/l, can be investigated accurately. The registration system may contain the exciter to excite the labels. The exciter can be a source of exposure. The exciter may also be a device for generating electromagnetic fields.

The first soluble layer may have a thickness in a range with a lower limit of 0.1 μm, preferably 1 μm and an upper limit of 150 m is m, more preferably 50 μm, even more preferably 15 μm. Mentioned thickness in this application may be the average thickness of the layer. The thickness of the layer depends on the porosity of the layer, type of analysis, the dissolution rate and the speed of the transport fluid (passive transport by diffusion or active transport using excitation). In the case of more porous layers is more efficient to use a thicker layer than in the case of less porous layers in order to obtain enough material of the reagent on the surface for biosensor analysis. In the case of thicker layers more efficient to use active transport to accelerate the achievement of the reagent to the surface of the sensor. In the case of very soluble thin layers, the probability of collisions label-surface advantageously rises relative collision label-the label in the liquid. Reducing the number of collisions label-the label reduces the frequency of occurrence of the aggregation of tags under test, which can improve the quantitative accuracy and reproducibility of the analysis marks the size of the nanoparticles.

The registration system may contain a matrix of sensor chips for the so-called touch multiplexing. Different sensors can be used, for example, in order to register different biological molecules to serve as positive or negative the m control or the purpose of calibration. Thanks to a very thin layer and short time of analysis, the reagents is not enough time in order to reach the neighboring sensors, which leads to the elimination or reduction of the problem of cross-reactivity or cross-contamination.

At least the first soluble layer can be uniform. Otherwise, it may be inhomogeneous, for example, consisting of Islands, bars, and other structures or patterns.

The layer can also be nonporous and/or porous, for better dissolution. An advantage of certain embodiments of the present invention in that it requires a small amount of reagent.

A sensor chip, in addition, may include a second layer for retention or immobilization exciting probes on the recording surface of sensor chip. This registration system makes possible the separation of labeled targets from unlabeled by capturing either related or unrelated to a recording surface of the factions. In working with the immobilized probes to capture also has the advantage of easier multiplexing (i.e. define different targets at the same time) due to their deposition on the sample.

Probes for capture can be designed to hold or Immobiliser and at least one label or the product of the interaction of at least one tag with at least one molecule of a target. Such retention or immobilization may occur on the recording surface of sensor chip.

A sensor chip, in addition, may include at least one of the calibration layer to provide reagents, calibration, provided that the calibration layer is soluble and is intended to provide calibration. The advantage that can thus be obtained an accurate recording system. In addition, the advantage is that the calibration system may be incorporated into the touch chip recording systems.

A sensor chip, in addition, may include at least one protective layer located over at least the first soluble layer, for protection, and a protective layer must be soluble.

This recording system makes it possible for long-term preservation of sensor chip, thereby reducing the number of non-working chips after storage. Multiple layers provide flexibility, for example, can be made of the buffer layer not containing biologically active substances to block the binding of any labels with biologically active substances in the manufacturing process, or a layer containing a calibration material or covering layer, acting as a separable protective layer against contamination, in the example, organic air pollutants emitted by the surrounding material of the cartridge during storage.

At least one label may be contained at least in the first soluble layer. An advantage of certain embodiments is that a sensor chip includes a limited number of different parts, thus reducing required in the production effort.

At least the first soluble layer recording system in the present invention may include soluble matrix containing one or more viscosity modulators, modulators of surface tension, regulators stickiness, pH regulators, blocking materials, thickeners, film-forming agents, stabilizing agents, buffers, detergents, gelling agents, fillers, destructive film agents, pigments, thixotropic agents, protective agents, or hydrating agents/solvents. Other soluble layers can also include such soluble matrix.

The recording system may include a sensor chip comprising at least one layer, which ensures rapid dissolution, to avoid aggregation of tags at least within the first layer, to preserve the biological activity of at least one label and interoperable at least one label with the target in optimalnyh or very favorable conditions.

The recording system in the present invention may include at least one reagent, which may constitute or contain an enzyme, coenzyme, cofactor, vitamin, mineral, the substrate of the enzyme or enzyme inhibitor. This recording system can include a sensor chip containing at least one layer, in turn, contains the reagents in order to ensure the interaction of at least one label with the analyte in the optimal or very favorable conditions. The above-mentioned enzyme may be the trigger.

The reagent may be adapted to reduce the loss of biochemical activity due to the collapse, shielding, protective coating or masking. The interaction of the at least one tag molecule-target can be achieved using any suitable methods of analysis, such as competitive analysis, response inhibition, the method of substitution, composite analysis, anticomplex analysis, immunological analysis, a method of clustering, analysis by hybridization or analysis method of blocking. An advantage of some embodiments of the present invention is the registration of analytes can be based on the dynamic behavior of the signal reception.

The registration system also may include himself sensor for determining the time of receipt of the sample on the recording surface. The advantage of this alternative implementation is that the registration of analytes can be based on the dynamic behavior of the signal reception.

In addition, the recording system may include a sensor designed to measure the volume of the liquid sample or part thereof. This registration system provides accurate, such as quantitative registration of a target molecule, which became a registered mark after interaction with the label.

In addition, the recording system may include a sensor designed to measure the local density of at least one label. This system provides accurate, such as quantitative registration of a target molecule, which became a registered mark after interaction with the label.

At least one molecule of a target may be a product of the enzymatic conversion.

The recording system may also include analysis tools for determining the amount of at least one of a target molecule. At least one molecule of a target may indicate the presence or absence of the analyte in the sample. At least one molecule of a target may be similar to the analyte in the sample. The recording system can provide quantitative results. The latter can be obtained by comparing different signals registration, outbound is from sensor chip.

Analysis tools can include means for counting the enzymatic kinetics. An advantage of embodiments of the present invention that can be investigated enzymatic activity.

The recording surface may be porous surface. An advantage of embodiments of the present invention that the ratio of surface to volume may be increased.

At least one label may be specific to the target label. Specific for the target tag can contain specific for the target probe. Specific to the target probe can be a nucleotide sequence containing the sequence complementary to the sequence within the above-mentioned molecules. Specific to the target probe can be an antibody to the target.

At least one label may be associated with capture probes through molecular tag, for example through bitenova label capable of interacting with other molecular label, such as streptavidin label on the capture probes on the recording surface.

The present invention is also concerned with a method of registering at least one of a target molecule in a sample, where the method includes: a contact of the sample with a sensor chip that includes at least a first soluble layer containing a reagent, and about who has the interaction between at least the first soluble layer on the recording surface and the liquid sample, thus ensuring the interaction of at least one tag with at least one molecule of target and registration dependent label signal reception. The method also may include the initiation of at least one label, for example, by irradiation or by exposure to electromagnetic field on its physical properties. The method may also include the measurement signal of the sensor prior to interaction, in order to calibrate the sensitivity of the sensor chip. This calibration can be calibrated sensitivity specific to the label and/or calibration of the thickness of the layer of reagent. Furthermore, the method may include processing dependent on the label signal, for example to get the number or concentration of analyte present in the sample. The method also may include the separation of bound and unbound label.

In addition, the present invention relates to a sensor chip for registering at least one of a target molecule in the sample, and a sensor chip is designed to receive at least one label for the registration of marks; a sensor chip contains a recording surface having at least a first soluble layer containing a reagent; this at least the first layer provides the interaction of at least one label as the mini is the mind of a single molecule-target thus, allowing you to register the signal reception on the basis of the label. At least the first soluble layer may contain a label. A sensor chip can be disposable.

The present invention also relates to a kit of elements for reception of at least one of a target molecule in a sample, where the kit includes a sensor chip that is designed to receive at least one label for the registration of marks; a sensor chip contains a recording surface with at least one soluble layer containing a reagent; this at least the first soluble layer is designed to communicate at least one tag with at least one molecule of a target, thus allowing you to register irrespective of the label signal and a specified number of at least one of a target molecule in a buffer solution. A specified number of at least one of a target molecule in a buffer solution can serve as a positive control and/or reference. The kit can also include a buffer solution not containing at least one of a target molecule, acting as a negative control.

An advantage of some embodiments of the present invention that the chemicals used and their concentrations can be changed to optimize it takes is th biochemical process.

Separate and preferred aspects of the invention set out in the accompanying independent and dependent clauses. Features of the dependent claims can be combined with the features of the independent claims and the features of other dependent claims of necessity and not necessarily as expressly set out in these paragraphs.

The principles of the present invention allow the design of improved methods and devices for the registration of chemical, biological and/or biochemical particles.

Mentioned, and not only the properties, features and advantages of the present invention will become apparent from the subsequent detailed description provided in conjunction with the accompanying drawings, which show, for example, the principles of the invention. This description is given only for the sake of example, without limiting the scope of the invention. Figures referenced below are located on the attached drawings.

Brief description of drawings

Figure 1 - schematic illustration of the recording system according to a separate embodiment of the first aspect of the present invention.

Figure 2 - schematic representation of a biosensor chip with soluble reagent layer for recording, suitable for a recording system based on the labels, according to a separate embodiment of the first aspect of the present image is etenia.

Figure 3 - schematic representation of a biosensor chip with soluble reagent layer for recording, suitable for a recording system based on the labels, according to another specific embodiment of the first aspect of the present invention.

4 is a graph of changes over time volumetric concentrations moving labels next to the recording surface (C_l,m,s) as a function of time obtained/valid registration method according to a separate embodiment of the second aspect of the present invention.

5 is a graph of changes over time of the local concentration connected/connecting the marks on the sensor surface (C_l,b,s) as a function of time obtained/valid registration method according to another specific embodiment of the second aspect of the present invention.

6 is a diagram of the method for detecting target molecules in the sample according to the present invention.

Fig.7 shows the results of the competitive analysis of morphine in the method according to the present invention.

Fig shows the effect of surface-active substances in the drying buffer at a competitive analysis of morphine, based on the present invention.

On the other figures the same refer to footnotes refer to the same or similar elements.

Detailed description of embodiments is subramania

The present invention will be described from the point of view of the individual embodiments and with reference to certain drawings but the invention is not limited, but only by the claims. Any reference signs in the claims should not be interpreted as limiting the scope of the invention. Described drawings - only schematic and not restrictive. The size of some elements in the drawings may be exaggerated and shown not to scale for illustrative purposes. When in the present description and in the claims, the term "containing", it does not exclude other elements or steps. Where to use a noun in the singular, refers to an indefinite number of the noun, unless otherwise indicated.

In addition, the terms "first", "second", "third" and others in the description and the claims are used for distinguishing between similar elements and not necessarily for describing the order or chronological order. It should be understood that such terms are interchangeable under certain conditions, and that embodiments of the invention described here, will allow no such sequences, as here described or illustrated.

Moreover, the terms "top", "above" and the like in the description and requirements of the s are used for descriptive purposes and not necessarily for describing relative location. It should be understood that such terms are interchangeable under certain conditions, and that embodiments of the invention described here, can not work with this spatial arrangement, as herein described or illustrated.

Subsequent terms or definitions are given solely in order to help understanding of the invention. These definitions should not be interpreted as having less than clear to the average person.

The term "sample"used herein refers to the mixture, which may contain at least one of the investigated analyte. The sample is likely to be a liquid, such as water mixture. Consequently, the "sample" can be interpreted as "liquid sample".

The term "analyte", as used here, refers to the substance, the presence, absence or concentration of which require the determination according to the present invention. Typical analytes may include, without limitation, small organic molecules, metabolites, such as glucose or ethanol, proteins, peptides, nucleic acid fragments, low molecular weight drugs, antibiotics or medicines, molecules with regulatory effect in the enzymatic process, such as promoters, activators, inhibitors or cofactors; viruses, bacteria, cells, cellular components, spores, DNA, RNA, microorg the mechanisms and their parts and products, or any substance for which can be found in the binding sites, the binding elements or receptors (such as antibodies), or which has antimony or ontogeneticheskie region binding. The presence, absence or concentration of the analyte can be directly determined by evaluating the presence, absence or concentration of the analyte, or, in another embodiment, it is determined indirectly by evaluating the presence, absence or concentration of the target or a target molecule.

The term "substrate"used herein refers to a molecule or material that can undergo enzymatic conversion.

The term "target"or "molecule-target", as used here, refers to the substance, the presence, absence or concentration of which is determined in accordance with the present invention. The term "molecule-target" should be understood broadly and can refer to, for example, a single molecule can designate a cluster of molecules, may refer to a complex of molecules, can refer to a molecule that is embedded in another material such as the substrate, etc. of the Target and the analyte can be identical, or the target may indicate the presence or absence of the analyte. In particular, targets, such as proteins or DNA, can be a characteristic component or product of analytes such as viruses, bacteria and other organisms, and therefore, the light of Edelstahl about them. In those cases, when the registration includes enzymatic analysis, the goal may be the product of the enzymatic conversion of the substrate by the enzyme and, therefore, may indicate the amount of the substrate or on the activity of the enzyme. The target molecules can also be polymers, metal ions and low molecular weight organic substances, such as toxins, drugs and explosives, which the present invention is naturally not limited. In the process of enzymatic analysis target can be marked for the emission of signal reception, the target may be immobilized on a recording surface comprising a biologically active coating.

The term "label"as used here, refers to a molecule or material capable of emitting the signal reception. Generating a signal reception includes a signal change. Labels suitable for use in various systems and methods of registration relating to the present invention are numerous and are described in detail in this technical field. Such marks may be an optical label, a radioactive label, a magnetic label, etc. Labels can be straight marks, which can be directly registered by the sensor. Otherwise, the labels may be indirect, to become Registrar is the subject in the process of further development. Usually, the label used in the methods of the present invention is specific to the target label, that is capable of specific binding with the target. However, also provides that in cases where the target is in purified form, it is enough to associate the label with the target.

The term "probe"as used here, refers to a binding molecule that specifically binds to the molecule target. The probes provided in the context of the present invention include biologically active substances, such as whole antibodies, portions of antibodies, such as Fab'-fragments (antigennegative domains), single-chain variable fragments, single variable domains, the variable domains of the heavy chain (Variable heavy chain domains), antibody heavy chains, peptides, antigenic determinants (epitopes), membrane receptors, or any kind of receptor, or part of, the substrate is exciting mutant forms of the enzymes, whole molecule antigens (haptens) or antigenic fragments, oligopeptides, oligonucleotides, military, nucleic acid and/or mixtures thereof, capable of selective binding with potential molecule-target. Antibodies can be raised) to non-protein mixtures as well as to proteins to peptides. Typically, probes are members of immunoreactive pairs or pairs of binding jet is about the affinity. The nature of the probe will be determined by the nature of the detected target. Often the probe is developed on the basis of specific interaction with a target, such as, without limitation, communication antigen-antibody complementary to the nucleotide sequence, carbohydrate-lectin, a complementary peptide sequences, ligand-receptor, enzyme, coenzyme, enzyme-inhibitor, etc. Probes also include probes for capture" for immobilization of target and/or marked targets on the recording surface by acts of recognition or binding. Probes and probes for capture can be flagged. In cases where the molecule-target immobilized by binding to a capture probe, the resulting complex is referred to as the target is a fascinating complex. In cases where the label used in the devices and methods of the present invention is specific for the target, it can be guaranteed with the use of a specific target probe that is associated with the label. As a rule, in those cases when the target is a protein specific to the target probe can be an antibody to the target. In another embodiment, when the target is a nucleotide sequence specific to the target probe can be the complementary oligonucleotide sequence. The term "analog target", as used here, refers to codesto, which can communicate with the probe or capture probe is worse than the target itself. Similar targets used in the competitive analysis in cases where the target is determined on the basis of competition with similar targets, for example by competitive binding with the probe or probe to capture. In particular, the analogue of the target bound to the probe or probe for engagement with the reduced strength of binding compared to binding of the target with the probe or probe to capture.

According to the first aspect, the present invention provides a system of registration and/or determining the amount of at least one of the target molecules, and, consequently, of the analyte in the sample. This recording system can be, for example, a system for the registration of chemical, biological or biochemical particles than the present invention is not limited. The recording system is usually designed for use for at least one of sensor chip designed for receiving at least one tag to create a registration on the basis of the label. A sensor chip, in addition, has a recording surface which includes at least the first layer, the first layer should be soluble in order to provide at least one reagent to interact with at least one label to at least one m is the solution, thus allowing you to register the signal reception on the basis of the label. Soluble layer can be referred to as soluble reagent layer, although in addition to the reagent layer can also contain other components. The term "soluble layer"used herein may refer to a layer consisting of a soluble matrix and, optionally, labels, samples, labeled samples, targets and/or analogs of targets. When soluble layer is in contact with the liquid sample, the sample is likely to dramatically exposed mobile reagents, i.e. within a short time.

A schematic overview of the registration system 100, which includes mandatory and optional components, illustrated in figure 1. The registration system 100 suitable for reception and/or determining the amount of at least one of the target molecules and, thus, of the analyte in the sample. The system 100 suitable for registration, and, optionally, to determine the number of target molecules or, hence, of the analyte in the sample, whereby the preparation of the soluble layer containing the reagent on the recording surface of sensor chip allows you to quickly record label as a direct or indirect indicator of the presence or activity of the analyte. As shown in figure 1, the recording system 100 is adapted for use with at least one sensor chip 1. E and other additional or optional components of an illustrative recording system 100, such as shown in figure 1, will be described in more detail below. A sensor chip 1 will be described with reference to Figure 2 and Figure 3 as an illustration than a sensor chip 1 is not limited.

A sensor chip 1 may contain some components, such as the carrier chip 2 having a surface 3. At least part of the surface 3 can be designed to be a place of registration using the detector 30. In other words, the surface 3 may contain the recording surface 33, where registration can be carried on the basis of the label. The carrier chip 2 can be made of material that allows the carrier chip 2 to support the various components located in and/or on the carrier chip 2. It may include insulating material, although the present invention is not limited with this. Plastics, such as vinyl polymers, polyimides, polyesters and styrene, can provide the required structural properties. Since it is desirable that a sensor chip was suitable for mass production, it is possible to produce, for example, of a sufficiently elastic material, which can be laminating, however, giving the finished chip suitable rigidity. The carrier chip 2, therefore, may contain a flexible organic material such as a polymeric material, such as polyester, in particular in comtemporary polyester materials Naftalan polyethylene and polyimide, or a composition of two or more of them. Another particularly desirable material, the chip carrier is an inorganic material, for example a semiconductor, such as silicon, or materials such as glass.

The touch surface 3 of the chip can usually contain the material of the carrier chip, which can bind a biologically active layer probes to capture target or analogues of targets, or a biologically active layer immobilized. The touch surface 3 of the chip 2 may be a porous surface in order to increase the ratio of surface to volume.

The term "biologically active coating" may refer to a layer of biologically active substances, such as probes for capture and/or target molecules, held or attached to a solid surface, such as a recording surface of sensor chip, and capable of binding or reacting with the target or labeled probe, respectively. Probes for capture and/or target molecules biologically active layer can be retained or immobilized on the surface by any known in this field of manner. These biologically active substances may be applied or attached to the recording surface in a site-specific manner, which means that the connection is enabled specific the Kie sites on these agents, for example, through impermeable to protein layer on the substrate. In another embodiment, surface3 may include a metal layer, for example a layer of a noble metal covering the chip carrier or its part.

In accordance with the variants of implementation of the present invention, the surface 3 of the carrier chip 2 includes at least one soluble layer containing a reagent, also referred to as soluble reagent layer 5, involved in the registration of the analyte on the basis of the label. Such a layer may be a dry layer. Usually at least one reagent layer 5 of sensor chip 1 may contain reagents chemical or biochemical nature to react with the target to form the signal reception, signifying the presence of the analyte in the sample. The term "reagent"as used here, refers to chemical, biological or biochemical reagent that reacts with the analyte and/or with the target in order to produce a recorded signal signifying the presence of the analyte in the sample. Suitable reagents for use in various recording systems and methods of the present invention include a number of active ingredients, selected with the purpose of determining the presence and/or concentration of various analytes. Within this know-how desirable the selection of the appropriate reagents. As is well known in danniebelle, there are many chemicals available for use with each of the different goals. Their selection is made against the designated target.

The reagent may include, for example, an enzyme, a coenzyme, an enzyme inhibitor, the substrate of the enzyme, cofactor, such as ATP, NADH, etc. to facilitate enzymatic transformations, vitamin, mineral, than the present invention is not limited. For example, in one preferred embodiment, a sensor chip in the present invention may include one or more enzymes, coenzymes and cofactors, which can be selected in order to determine the presence of metabolites or small molecules in the sample. In addition, at least the first layer of sensor chip 1 may also contain labels, buffer salts, detergents, sugar, etc.

At least a first reagent layer 5 may be a thin layer. At least a first reagent layer 5 must be thin enough that the liquid sample was quickly hydrational or dissolved, a thin layer of reagent 5. It is desirable that at least the first reagent layer 5 has a thickness in a range with a lower bound of 0.1 μm, preferably 1 μm, and the upper border of 150 μm, more preferably 50 μm, even more preferably 15 μm. The thickness used in this invention may have an average layer thickness. The reagent layer can be effectively homogeneous, or it may include patterns or patterns, such as a structure in the form of Islands or strips, or may alternatively, or additionally, be porous for better solubility. Further, the thickness refers to the average thickness of the material in the patterns, structures, Islands, strips, etc. a Layer of reagent can also be porous for a better solution, for example, it may be microporous or nanoporous layer. In the production process, the reagent layer may be, for example, deposited on a cooled substrate to reduce interaction between the reagent layer and the recording surface at the time of production. The reagent layer, for example, can also be deposited using lyophilization. In a preferred embodiment, the label 6 is contained in the reagent layer 5 and diffuse through and into the reagent layer 5 to a recording surface 33 when the last contact with the liquid sample. Marks 6 will be at a small distance for diffusion through a thin layer of the reagent, therefore, diffusion on the recording surface will occur quickly. In addition, the capture efficiency will be greater in the case of subtle than in the case of a thick layer. A thick layer will need more time, so that the liquid sample has hydrational or melted it, and the longer it will need reagent or label 6 to reach the ü recording surface 33. This may increase the time required to determine the concentration of the analyte and enter in the definition of the error. When using relatively thick layer, it is advantageous to use active transport to accelerate the achievement of the reagent to the surface of the sensor.

For purposes of illustration and not limiting theoretically embodiments of the present invention, a suitable thickness of soluble reagent layer 5 can be determined on the basis of the law of diffusion. Quick dissolving and diffusion of reagents - this is the process, having the character of spike. If diffusion is the primary mechanism of transport (i.e. active transport is not used), a suitable layer thickness ofLcan be approximately computed as follows:L≈(Dt)^1/2.

WhereD- constant diffusion of the reagent dissolved in the layer,t- the desired reaction time. In other words, a suitable layer thickness ofLcan be approximately computed as the square root works constants of diffusion of the reagentDdissolved layer and the desired reaction timet. The diffusion constantDas a rule, will be different for different (bio)chemical substances.

IfDorder 10^-10m²/s when the reagent is released into the liquid, for example, in the form of a small protein, acting as a substrate for the enzyme, and the time required to implement the tion is 1, the suitable thickness of the layer is approximately equal to 10 μm. Much more than a thin layer will give a strong time dependence of the concentration of the reagent in the reaction. Much thicker layer consumes an unnecessarily large amount of reagent and, in addition, creates a distribution of target molecules distant from the recording surface. When the reagent in the reagent layer 5 is associated with a larger object, for example, nanoparticle size 300 nm,Dis of the order of 10^-12m²/s At the required reaction time of 10 with a suitable layer thickness is about 3 μm. In the embodiment, in which the labels 6 are contained in the reagent layer 5, it is possible to make a similar calculation. If diffusion is the main mechanism of transport, duration of surge or diffusion, thus, we can approximately be calculated asT_b=L²/DwhereL- layer thickness (in meters),D- constant diffusion labels in the fluid (m²/s), andT_b- time diffusion layer, in accordance with what can be neglected enhanced friction that occurs due to the presence of the surface. For example, labels such as nanoparticles with a diameter of 300 nm, in a liquid water-based can normally have a constant diffusionDorder 10^-12m²/C. When the layer thickness ofLequal to 3.2 mm, estimated time of diffusionT_bextending t is about 10 C. Hence it becomes obvious that the burst duration Tb depends on the square of the distance, which gives special importance to the use of ultra-thin layers, if necessary, to reduce the time of registration. Speed test also limited by the rate of binding of the target and sample capture. For a given capture probe, such as an antibody, a probability ofpthat formed the target is a fascinating complex, for example, communication antigen-antibody, increases linearly with timetin the limitp<1. The increase in the probability per unit of timedp/dtshown in equation

dp/dt=k_on[T],

wherek_onthe constant Association for binding of a target molecule with a sample capture (in l/(mol×C)),Tthe concentration of the target in the liquid (mol/l). For example,k_on= 10⁵l/mol×for communication with the drug-antibody, and [T]= 100 nmol/l givedp/dt= 0,01 s^-1. This means that when the above parameters, the complex target-invader is formed through 10 with a probability of 10%.

At least one soluble reagent layer 5 of sensor chip 1 may include soluble matrix 7 or dried porous material containing reagents and/or labels and/or the number of adjuvants to enhance the properties or characteristics of the reagent, and/or protective agents for preservation of components inside R is starimage layer 5 during processing and storage. Matrix 7, as a rule, can produce rapid dissolution, to avoid sticking labels and retain the biological activity of substances. The chemical composition may include substances that are needed to facilitate the placing of the mixture of the reagent on the surface 3 of the carrier chip 2, and in order to improve its adhesion to the surface of the chip 3, or to increase the rate of hydrogenation of the mixture of the reagent with the liquid sample. In addition, the reagent layer 5 may contain features that are selected to enhance the physical properties of the resulting dried layer of the reagent and the absorption liquid sample for analysis. Examples of support materials suitable for use in the mixture of the reagent include viscosity modulators, modulators of surface tension, the regulators of adhesion, pH regulators, blocking materials, sealants, binders, stabilizers, buffers, detergents, gelling agents, fillers, agents, exposing film, dyes, agents, providing thixotropy, silica gel, and agents that promote the dissolution and hydration.

It is desirable that a sensor chip 1 contained the excitation mechanism for the excitation of the liquid, which is provided for the reaction. Excitation favorably, for example, to increase the speed, accuracy, sensitivity and/or JV is civicnet test by mixing agent in the fluid, transporting the reagent registration and/or increasing its concentration, as well as with the severity of the test, which is achieved, for example, by cleaning or by application of electromagnetic voltage. Therefore, the excitation process preferably used for the preparation of reagents near the biosensor chip. A highly preferred method of excitation is based on the magnetic excitation. Magnetic excitation is possible if the probes or other compounds that play a role in the observed reaction, associated with a magnetic particle, and/or when areagirls magnetic particles added to the reaction chamber. Therefore, in a highly preferred embodiment, a sensor chip 1 and/or reading system includes at least one component of magnetic excitation, such as an electromagnet or conductors, for creating an electromagnetic field near the surface of the sensor. In the case of magnetic excitation, registration, however, can be done by magnetic and/or optical registration with the use of additional optical labels. This is described in more detail below.

A sensor chip 1, as a rule, can take at least one label for registration on the basis of marks after interaction with molecules of the th target. In a preferred embodiment, the label may be at least in the first soluble layer. Otherwise, the label can be attached to the sensor surface as a so-called second soluble layer. Labels can also be delivered outside of sensor chip, for example, if a water-based liquid contains a label in the appropriate concentration, or as a component of a liquid sample. Labels can also be delivered from another location in the chamber of the Desk or the cartridge, preferably under the control of the excitation mechanism. Label(s) is(s) to interact with the molecule-target, which usually can be done by linking. By using a labeled binding molecules, each act of binding or registration typically generates the signal reception and indicates the presence or absence of activity of a target molecule. Linking and neisvaziuosiu ways, as provided in the present invention include immunological analysis, DNA hybridization, and analysis-based receptors, which are widely used in the medical community as a diagnostic test for a wide range of target molecules. Applicable methods of analysis include sandwich-analysis, anticomplex analysis, and analysis using blocking agents, review the th for example "the Immunoassay Handbook", published by Elsevier Science edited by David Wilde. In sandwich analysis using drip probes 61 label 6 usually keep close to the recording surface, for example, due to electrical and/or magnetic fields to attach drip probes 61 to the recording surface 33 for the formation of structures of molecular sandwiches" in the process of interaction of the target with drip probes 61 and the recording surface 33. Various types of (non)binding assays can employ optical label, such as, for example, fluorescent, chromogenic, scattering, absorbing, refracting, reflecting, SERRS-active or (bio)chemiluminescent labels, molecular beacons can use a radioactive label, you can use an enzymatic label, or may use magnetic particles as labels. Optical labels, as a rule, can emit light detectable by the detector, for example, in the visible, infrared or ultraviolet wave range. However, the invention is not limited to, optical labels in this application may refer to labels, radiant in any suitable and recorded wave range of the electromagnetic spectrum. Magnetic marks provided in the context of the present invention include, without limitation, metal is static or magnetic particles, or nanoparticles. Magnetic label may contain any suitable form one or more magnetic particles, for example, magnetic, diamagnetic, paramagnetic, super-paramagnetic, ferromagnetic, with any form of magnetism that creates a magnetic moment in a magnetic field, either permanently or temporarily. An example of a suitable material magnetic labels may be particles of Fe₃O₄. The size of the magnetic labels are not crucial in most embodiments, but for many applications of biosensors is highly desirable that the marks were small. The preferred magnetic labels can be generally expressed the greatest diameter in the range from 5 to 5000 nm, preferably from 10 to 2000 nm, more preferably from 20 to 1000 nm, even more preferably from 50 to 500 nm. Check magnetic label is typically produced by the application of electrical, magnetic or electromagnetic field and the use of magnetic or non-magnetic sensor. The label refers to molecules or materials, ecovalence associated with the sample, unless otherwise specified. A label can be attached to the sample probe to capture the substrate, the target or analyte, preferably by covalent bonds, although other types of connections, such as hydrogen bonds, are also possible. Depending on the type of the performed analysis, the marked moleculesin or contact with stationary probes for capture (sandwich assay), or compete with counterparts targets for the probes to capture (competitive analysis). After removal of the excess (unbound) labels, the number of related tags is measured. Thus, linking the types of analysis may typically involve the adhesion of labeled binding molecules with the solid substrate in quantities reflecting the concentration or presence of the target. In another embodiment, a labeled binding molecule, such as a labeled analogs of the targets may be associated with the recording surface and the displacement of labeled analogs of the target by target molecules can lead to the reduction in the number of labels near the recording surface. Described a large number of variants of methodologies linking analysis and all of them are included in the scope of the present invention.

In the sensor chip 1, for example, may on its recording surface, can be coated with additional layers, for example, a layer that performs the role of a protective layer, calibration layer or buffer layer. The protective layer can protect the individual parts of sensor chip 1 from internal or external chemical or mechanical influences. The calibration layer can be used for calibration of sensor chip with respect to the applicable specific labels, thickness of layers, etc. Can, for example, to add a known amount of the target molecules and the like, similar to the target molecules in a soluble layer on the sensor chip to achieve this calibration. The latter, therefore, allows nutricional calibration. In addition, there may be applied a layer for a positive test or a negative test. Such layers can usually provide the necessary components for validation of sensor chip, causing a clear positive or clearly negative response, allowing, thus, the quality control of sensor chip 1. The buffer layer typically provides buffering between two different layers that do not have or initially should not interact with each other. Detailed examples of such layers will be discussed in more detail in a different implementation.

The liquid sample, which must be in contact with the recording surface, can cause the hydrophilic/hydrophobic forces on the wetted recording surface 33. Additional protective layer can be applied for protection from the forces introduced by the liquid sample.

To register signal on the basis of marks in the recording system 100 may be included, in addition, at least one detector 30 for registration marks on the surface of sensor chip 1 or close to it. Such a detector 30 recording system 100 may, therefore, be able to register the marks or, more precisely, the answer to their excitement on the recording surface 33. The detective who R 30 may be embedded in the carrier chip 2, as a result, thus, can be obtained biosensor technology "lab-on-chip", or, in another embodiment, it may be built into the registration system 100, adapted to include a touch of the chip 1. Otherwise, the registration system may be a standalone system, placed in a desired position for registration on the recording surface of the carrier chip. In the case of "lab-on-chip active elements of the detector 30 can be located on the carrier chip 2 touch the chip, and the registered signal can be converted and transferred to the external in relation to the sensor chip 1 card reader. In another embodiment, the active elements of the detector 30 can be located in the registration system 100 outside of sensor chip. The detector 30 in this case is intended to record the signal recording surface 33. For example, when optical detection of the latter can be achieved by focusing on the recording surface 33 and the data collection.

At least one detector 30 may be any suitable detector, for example, an optical detector for optical registration marks, such as optical or magnetic label and/or a magnetic detector for registration of magnetic labels. An optical detector for reception of optical signals, for example luminescent signals from optical labels or indirect fluorescent labels, magnetic labels, may be a photodetector, a charge-coupled device (CCD), the device with the injection of charge (FDI), complementary metal-oxide-conductor (CMOS), photomultiplier tube, avalanche photodiode, a semiconductor optical device registration, a microscope or video camera. At least one detector 30 may be a number of detectors for the recording of various ray fluorescent radiation collected from the sensor chip 1. At least one detector 30 can be a multipixel detector or a number of many single-pixel detectors. Such a detector may be, for example, a charge coupled device (CCD) or a device with the injection of charge (FDI), near the photon multipliers, near avalanche photodiodes or other radiation detector, comprising a set of individual detector pixels. It is desirable that the width of the at least one detector 30 or, in the case of application of multipixel detectors, detector element of the detector 30, was such that the registration could be carried out on all the sensor chip 1 or the touch chips or spatial certain areas on the sensor chip 1, with certain spatial region is such, that almost always no more than one strip or spot reagent layer was within the zone, recorded some of the pixel from the scan to determine if there are any stripe or spot on the layer(s) of reagent 5 registration marks during contact with the liquid sample.

At least one detector 30 may also be a magnetic detector such as, for example, the detector on the basis of the hall resistance or magneto-resistive detector, such as, for example, AMR (anisotropic magnetoresistity) detector, Supermagnete-resistant detector, or a TMR (tunneling magneto-resistant) detector. Magnetic sensor elements on the basis of other principles, such as SQUIDS (superconducting quantum interferometers), may also be used in the claimed recording system 100. The detector 30 may also be based on other principles of registration of magnetic particles and, thus, can also be a biosensor based on force sensor (force-amplified biosensor), cantilever force sensor, microbalance, gauge, impedance, or atomic force microscope, which are defined by the forces emanating from the magnetic particles or magnetic particles.

Registration of magnetic particles can also be carried out on the basis of optical phenomena such as refraction, absorption, scattering, fluorescence, etc. Thus, the detector 30 may be an optical detector for registration of magnetic particles. Typically, the detector 30 can be bound with direktorijum device 32 for movement of the detector 30. Detectoradware device 32 can usually be designed to control the detector 30. Generally, it can be placed outside of the carrier chip 2, and even out of touch chip and can be connected to the detector 30. However, it can also be included in the carrier chip 2, or in the sensor chip 1. The detector may be adapted, for example, by installing in the correct position or the provision of additional focusing elements to check labels or outgoing signals registration on the recording surface 33.

The recording system 100, in addition, may contain the exciter 31 intended for the excitation of used tags. Depending on what markers are used, the exciter 31 may be, for example, the optical unit for excitation or magnetic device for excitation. The exciter 31 can be controlled by the device for moving exciter 34. It can probably be located outside of the carrier chip 2, and even outside of sensor chip 1 and can be connected to the excitation device. However, it can also be included in the carrier chip or a sensor chip 1. Device optical excitation can represent, for example, the irradiation plant that includes one or bol is e radiation sources generating a beam for irradiation of the sample on the sensor chip containing optical labels. At least one device for irradiating 31 may be any suitable for use in a system for optical detection of a source of radiation, such as light source. A device for the irradiation of 31 may also contain a source of white light that can be filtered on a few rays with a specific wavelength or in a specific wave range. A device for the irradiation of 31 may also contain one or more monochromatic optical sources such as lasers. A device for the irradiation of 31 may include argon lasers, diode lasers, helium lasers, dye lasers, titanium-sapphire lasers, YAG-neodymium lasers (lasers YAG garnet with neodymium) and other Device for the irradiation of 31 may contain custom radiation source, such as, for example, the configurable semiconductor laser, to sequentially generate at least one beam, or at least one semiconductor laser for simultaneous or sequential generation of one or more beams. You can use a variety of irradiation devices 31 to create the possibility of multiplexing. Irradiation plant can be designed to generate electromagnetic what about the radiation, suitable for excitation of labels. For example, if the generated radiation is fluorescent radiation, the length of the optical wavelength of the exciting radiation can typically be, for example, in the range from 200 to 2000 nm, or for example, in the range from 400 to 1100 nm, without limiting the present invention. Such installation can be embedded in the carrier chip 2, or may be external to it; it can also be integrated in the sensor chip 1 or be external to it. Magnetic device for excitation, for example, be an electromagnetic unit for generating an electromagnetic field for applying an electric or magnetic field to the sample containing magnetic label with the target orientation of the magnetic particles. Magnetic device for excitation 31 may be, for example, a magnetic field generator that generates a magnetic field for magnetizing and orientation of magnetic labels. Magnetic device for excitation can be embedded in the carrier chip 2, or may be external to it, also it can be integrated in the sensor chip 1 or be external to it. Magnetic device for excitation can be an electromagnet, the coil winding without core, straight wire, a guide, a permanent MAGN is t, the coil inductance. It can be an external magnetic device for excitation or may be combined with the carrier chip 2.

As mentioned above, the detector 30, a device for excitation of 31 and/or moving their devices 32, 34 can optionally be external to the sensor chip, or even external to the cartridge 50 containing a sensor chip 1 and the device for feeding the liquid sample 20 and/or a device for containing the liquid sample 11, or a device for containing the test fluid 12. If the detector 30 or the device for excitation 31 are out of the cartridge 50, the cartridge 50 can be provided by Windows, so that the recording device 30 could register in the sample, etc.

Signals indicating the registration can be delivered on a schema for analysis, which can be used to perform any of the following analysis algorithms of the present invention.

In addition, the recording system 100 can include a schema for analysis 40, intended for processing reception signals, or signals corresponding to them. Generally, it can be designed to perform a set of algorithms for processing received from the detector results. Scheme for analysis 40 may be PR is naznachen to determine the concentration or distribution of the analyte in the sample and/or for processing the received signals from the detector, for example, for determination of enzyme activity. In addition, the scheme for analysis traditionally includes the connection with the detector device 30 for the evaluation of signal reception, the corresponding concentration of the target. The concentration of the analyte can be calculated by comparing the reception signals coming from sensor chip, for example, in different moments of time while providing a sample of sensor chip 1. Scheme for analysis 40 may provide a digital binary value indicating whether the marked target. The system 100, and specifically the schema for analysis 40 may, in addition, carry out statistical processing of the results of the Desk, for example, to compare two different measurements to verify, did loosely coupled marks for registration. Scheme for analysis 40 may also include a device, you need to determine that the sample was received by the sensor and that the amount of sample is sufficient for testing. Scheme for analysis 40 may include a treatment device 42, such as, for example, a microprocessor and/or memory element for storing the received and/or processed information about the assessment. Additionally, there may be used conventional input devices-output. Scheme for analysis 40 can be controlled by using appropriate software respecialization equipment to perform the stages of assessment. Scheme for analysis 40 may, therefore, be implemented in any suitable way, for example, using specialized equipment, or by using appropriately programmed computer, microcontroller or embedded processor such as a microprocessor, a programmable matrix of logic elements, such as PAL, PLA or FPGA or similar. Scheme for the analysis of 40, as a rule, can store and display the analysis results on any suitable external device 44, such as a visual display device, image recording device, printer and so on, or in another embodiment may transmit data to a separate device. Scheme for analysis 40 may also be connected with a local area network or a global computer network for the transfer of results to a remote point. Scheme for analysis 40 may at least partially be in the cartridge 50, or if desired, may be located outside of the cartridge 50. Scheme for analysis 40 may be connected with the cartridge 50 with the appropriate contacts on the surface of the cartridge, for example, using a terminal.

In addition, the recording system 100 can include a device 10 for holding liquids, for example liquid sample, which can act in one or more containers in the role of a source is in resources for the recording system. Such a device for fluid 10 may be a device 11 is designed for the maintenance of the investigated liquid sample, i.e. a specialized source 11 of the sample, which presumably contains the analyte. The device 10 for the liquid content may also optionally contain at least one device 12 intended for content control sample, such as a sample containing a predetermined concentration of the analyte or target employees, for example, as a positive control or as a reference sample and/or sample not containing the investigated analyte or target, for example, "form" or the negative control. The sample is most likely a liquid sample. The aqueous mixture is very suitable for use in this registration system. If the label is provided separately, the recording system may optionally include at least one source of labels, not shown in figure 1.

The recording system 100 can also include a device for delivery of a sample of 20 from the device to the contents of the sample to the sensor chip 1, for example, for the touch of the chip 1 with the liquid sample. Device for delivery of the sample 20 can include gravimetric power fluid and may also include in the BOJ system of tubes and valves, for example selectable and adjustable valves, to ensure the delivery of fluids from the device 11 to the liquid content of the sample and the device 12 to the content of the control sample to the sensor chip 1. In another embodiment, fluid can actively or passively vykazyvaetsya of the devices 11, 12 to the sensor chip 1. The above system elements may be located on the cartridge 50, such as a disposable cartridge 50. You can also use the drive circuit 22 to the control device 20 for shipping the specimen.

The recording system 100 may, but is not obligated, to include, in addition, to facilitate the dissolution of the device 60, in order to promote the dissolution of a soluble layer(s) and the diffusion of dissolved components on the sensor chip 1 or close to it. To facilitate the dissolution of the device 60 may include a magnetic device for excitation, heating element, or any other mechanical or acoustic means to facilitate dissolution and diffusion.

The recording system 100 may, but is not obligated, to include a device for controlling a temperature of 62, in order to ensure the appropriate temperature on the sensor chip 1 or close to it. Device for temperature control 62 may include heating and/or cooling element, p is Solea thus to control the temperature of sensor chip 1, or liquid sample held in the sensor chip 1. Device for temperature control 62 may be external or internal to the sensor chip 1 and the external or internal to the cartridge 50. You can use different ways of heating and cooling elements, comfortable inside of the recording systems, including, without limitation, electric heaters, resistive heaters, thermoelectric heaters and coolers (Peltier device), radio frequency heaters capacitively coupled, heat-absorbing devices, heaters for liquid schemes, heat, chemical heaters, and other types of devices. In some embodiments, the implementation of the liquid inside the recording system 100 is heated by an external heating mechanism. You can also apply heat via radiation. Device for temperature control may also include a temperature sensor for detecting temperature of the recording surface 33 or the temperature of the sample near the recording surface 33.

In the recording system 100 may include a cleaning device 64 for cleaning the recording system or its parts, such as, for example, the sensor chip 1 or the recording poverhnosti in the sensor chip 1. Cleanability register what her system 100 typically depends on the presence of smooth surfaces. Surface in contact with biologically active compounds preferably can be smooth, accessible for cleaning solutions and legkovushkoj. The choice of materials for the parts of the recording system, which must be resistant to a wide range of pH levels, allows the use of standard cleaning solutions for biological materials, often with extreme pH values. Cleanability of surfaces also includes their sterilizeable. In the case of planar systems, sterilization by ultraviolet partly suitable, but assumes a corresponding system stability to ultraviolet radiation. The choice of UV resistant materials comprising a sensor chip 1, allows to apply the processing with ultraviolet radiation for sterilization before use.

In the recording system 100, in addition, optionally, may include a sensor for measuring the volume and surface density of the liquid containing the labels, on the surface of the biosensor. The latter can be done by optical measurement, pressure measurement, measurement of volume in the device 10 for delivery of the liquid, or by detecting the presence of fluid and/or fluid flow in various parts of the delivery device of the liquid 20. The time of delivery of liquid to the surface of the sensor signal is to be determined, for example, using changes in capacitance or temperature. The latter can provide additional information about the time response of fluorescent labels and/or they can be to serve as a contribution to the management of the detector 30, a stimulating device 31 or in the response signal to the controller 22 for controlling the device 20 for delivery of the liquid. Time of delivery of the liquid on the recording surface 33 may also be taken into account by the scheme for the analysis of 40.

You can also use the agent that is designed to further stimulate the interaction of at least one tag with at least one molecule of a target. This pathogen can be magnetic, electric or acoustic field. It can improve the dynamic performance analysis, increasing the intensity of the binding. The pathogen can also be applied to the removal processes or improve accuracy, in order to increase the specificity and sensitivity of analysis.

A sensor chip 1 may contain a matrix of sensors, for a so-called touch multiplexing. Various sensors can be used for registration of various biological molecules, can be used as positive or negative control or can be used in calibration purposes. In modern biosensors different sensors the usual exposed to the same chemicals. This creates problems of cross-reactivity or cross-contamination, for example due to the fact that the same label can contact with different surfaces of the sensor. Thanks to the limited number of reagents, due to the fact that the reagent layer, as a rule, can be subtle and low time of the analysis, in some embodiments, implementation of the present invention the reagents may be sufficient time in order to reach the neighboring sensors on the substrate. The path will be approximately (Dt)^1/2whereDis the diffusion coefficient,t- time. Due to short reagents, thus, do not reach the neighboring sensors, which eliminates the problem of cross-reactivity and cross-contamination. This increases the capacity of the multiplex biosensor.

The present invention thus also provides for the screening sample. Non-limiting examples of screening tests may be given by the following. The number of sensors is supplied with reagents, characterized by the fact that these reagents differ in missing in each of them (bio)chemical component, and the fact that the sensor gives a positive signal when the sample provides the missing component, and the fact that the sensor gives a negative signal when the sample does not contain the missing component. Thus, the sample m which can be checked for example, the presence of cofactors.

Further embodiments of and examples according to the first aspect is provided below.

According to the second aspect, the present invention provides a method of registering at least one of a target molecule in the sample. This method usually allows you to count the number of target molecules in the sample. This method of registration of the target usually involves contact of the sample with the sensor chip 1, containing at least one layer with a reagent. Typically, the method also includes providing interaction between the at least first layer of the reagent and the liquid sample, which leads to the dissolution of the first layer of the reagent and the interaction of at least one tag with at least one molecule of a target. The latter is depicted in the example of figure 6, showing the diagram of a method 200 according to the second aspect of the present invention, illustrating both established and optional stages of an exemplary method of registration.

At the first stage 202, a sample and a sensor chip containing at least a first dissolvable reagent layer 5 are in contact with each other. Typically, this involves contacting the fluid sample, for example a drop of liquid sample, with sensor chip 1 by gravity or capillary forces. In another variant is NTE, the fluid sample may also be actively or passively connected to the sensor chip 1. Contact of the sample with the sensor chip 1, thus, may include management of feeder pattern.

In the second step 204, the method includes providing communication between at least a first dissolvable reagent layer 5 and the liquid sample, which leads to the dissolution of the first soluble reagent layer 5 and to the interaction of at least one label 6 with at least one molecule of a target. The reaction between at least a first soluble layer 5 and the liquid sample, therefore, is, as a rule, the reaction between soluble reagent layer 5 on the recording surface and the liquid sample. The latter, therefore, makes possible the emergence of the free reagent on/near the recording surface 33 of sensor chip 1. This usually makes possible the interaction of at least one label 6 with at least one molecule of a target. This method may also include providing communication between other soluble layers and the liquid sample, thus ensuring the implementation of the remaining phases of the interaction. Labels can be soluble in the reagent layer or other soluble layer, leading, thus, to the formation of free labels for wsimages is via at least one molecule of a target. Otherwise, the label may appear the other way, for example with the introduction of the liquid containing the labels, in the sensor chip 1. Typically, to provide an interaction between soluble layers and the liquid sample, the liquid meniscus of the sample must pass soluble layer. The latter probably takes much less time than the total time of the analysis. Typically, the wetting occurs when the passage of the meniscus of the liquid sample through the registering surface.

In the third step 206, the check signal reception on the basis of the label can be performed using any suitable method of registration in accordance with the selected labels. Provided appropriate ways of registration include optical registration, such as, without limitation, fluorescent registration and increased surface resonance Raman scattering (surface-enhanced resonance Raman scattering, SERRS), and how magnetic Desk, for example, using a Hall sensor, the effect of supermagnetosonic, tunnel magnetoresistive (TSR) or anisotropic magnetoresistive (AMR). Characteristically, this method can therefore include, as indicated by step 208, the excitation of the labels to create a signal from the tag needs to be defined. Depending on the form used is etok, such excitation may be a sample illumination or excitation pattern of the electromagnetic field to Orient the magnetic label. If the excitation is the stage of irradiation, it is usually used beam suitable, for example, the wavelength or intensity for excitation of optical labels used in the sensor chip 1. If the excitation is a stage of excitation of the electromagnetic field, the electromagnetic field is usually selected so as to cause orientation of magnetic labels, recorded magnetic sensor in the registration system.

The registration method 200 for registration and/or quantitative determination can also include the step of processing the signal reception on the basis of the label. The latter is illustrated in step 210. Processing may include obtaining qualitative or, more likely quantitative result from the signal reception on the basis of the label. Processing may be based on predefined algorithms using neural network or any other suitable means. Such processing can be automated.

In one embodiment, at the stage preceding the registration can be made optional phase branch 212, which is the separation of unbound or weakly bound label from the associated labels. Latter may be an advantage to achieve more accurate measurements, as it allows to avoid the influence of unbound or weakly bound labels on the results of registration. Removing unbound or weakly bound label can be guaranteed in the case of removal of unbound or weakly bound label from the associated labels associated with the recording surface through a biologically active substance or molecular tag. The latter can be accomplished by physical or chemical forces, such as field flow/flow, acoustic field, the gravitational force, the electromagnetic field, to move and/or remove unbound label. Approximate technique Department includes clearing, settling, centrifugation, sonication, the use of magnetic and/or electromagnetic fields and gradients/intensities of the field.

In some embodiments, the implementation phase separation associated labels from unbound may not be required. This can be achieved, for example, by providing a probe representing a molecular beacon, which contains the oligonucleotide sequence, complementary to the DNA sequence of the target and which is dual marked with pigment and quencher of luminescence (e.g., Dabcyl) at each of its two ends. In its operating condition the signal from the pigment is suppressed by the quencher of luminescence. When the complementary members shall etelnost it hybridises with the DNA target, the beacon is activated, and the signal can be registered. One example of molecular beacons can serve as beacons reinforced surface resonance Raman scattering, which are dual labeled probes with different colors on each of their two ends. The second pigment is specially designed so as to be capable immobilizative oligonucleotide probes on the recording surface, representing the appropriate metal surface. In the absence of DNA target, the beacon immobilized in the "off state" on the metal surface that allows you to register a range of enhanced surface resonance Raman scattering (SERRS), corresponding to both dyes. When the complementary sequence it hybridises with the DNA target, the beacon is activated and one of the pigments is removed from the surface. This leads to the change of the SERRS signal. In another separate embodiment, which does not require separation of bound and unbound label can be represented by at least two fluorescent labels, forming a pair of resonance energy transfer fluorescence (CHEF), and one tag associated with the mark registration and the other is movable in the fluid. Only when the two labels are in close proximity to others is g to each other, flyuorestsiruyut at the Raman wavelength CHEF, the couple is registered.

The registration method 200 can also include a stage of calibration. Therefore, the touch signal may be measured before wetting after wetting, in order to calibrate the sensitivity of the sensor chip to the label and/or the layer thickness of the reagent. This calibration step can be performed using predetermined algorithms, and the results of this calibration step can be taken into account at the stage of signal processing, for example, when calculating the results of the registration.

The registration method 200, in which the solution pre-incubated with magnetic particles according to the present invention, can be combined with magnetic extraction and analysis method of capture. Immediately after wetting the particles are transferred to the surface of the sensor. Splash reagent can strengthen the registration process, for example, by local security biochemical conditions (e.g. pH, salts, organic molecules), optimize for speed, sensitivity and specificity (don't)bind to the surface of the sensor.

According to a third aspect, the present invention provides a sensor chip 1 to the registration of one or more analytes in the sample. More specifically, a sensor chip 1 is provided for the use of the recording system 100, as explained above. A sensor chip 1 to the registration and/or counting the number of at least one of a target molecule in the sample can be designed to contain at least one label 6 to allow registration on the basis of the label, as well as to contain the recording surface 33 to provide at least a first soluble layer. The first soluble layer 5 typically includes at least one reagent and, as a rule, can also be called soluble reagent layer 5. Soluble reagent layer 5, usually located on the recording surface 33. When soluble reagent layer 5 is provided in contact with the liquid sample, it usually allows at least one label 6 to interact with at least one molecule of a target, thus creating the possibility of signal registration registration on the basis of the label. Other characteristics, features and benefits, probably the same as described for the sensor chip in the first aspect of the present invention.

According to a fourth aspect, the present invention provides a kit comprising at least one touch chip, similar to those described in the first aspect of the present invention, in combination with a number, for example, predetermined, at least one molecule m is Sheni in the buffer solution. The latter can serve as a positive control and/or reference. Select the molecule-target, or select a target molecule in a buffer solution usually depends on the type conducted by the touch chip analysis. Such a sensor chip 1, for example, is suitable for the analysis of the enzymatic activity of an enzyme that can convert a substrate of the enzyme, introduced in a reagent layer on the sensor chip, although the present invention is not limited to this only and can be used in any suitable analysis methods, described in any of the aspects of the present invention and/or in any of the presented options, implementation and/or examples. In addition, such a kit (optional) includes other components, such as a predefined number of control fluid involving the measurement of the negative control, in which a sensor chip must specifically to provide a negative answer to the Desk, showing the absence of the registered target molecules. Various aspects of the present invention will now be illustrated by a number of individual embodiments and examples that the present invention is not limited.

In the first separate the embodiment presents the recording system described above for the first aspect, and the recording system 100 prex is assigned for use with at least one sensor chip 1, a sensor chip 1 includes a first soluble reagent layer 5 and the second soluble layer. Partly referring to Figure 2, the preferred implementation of sensor chip 1 are presented in accordance with the present invention. A sensor chip 1 includes a carrier chip 2 having a surface 3 with the recording surface 33, and the first soluble layer of the reagent includes a soluble matrix 7 and at least one label 6. Mark 6 provided with a probe 61. Suitable first soluble reagent layer 5 can usually lean on the recording surface 33, providing the proximity of the label to the recording surface 33, reducing, thus, the time of registration. This is the first soluble layer 5 can be brought into contact with the liquid sample corresponding devices, for example, by gravity or capillary action, or, selectively, using compression, or under a vacuum. Recording surface 33 of sensor chip 1 in this embodiment is covered with the second layer 4, a biologically active layer, for example, containing the capture probes 41, such as antibodies or oligonucleotides, target or similar targets. The carrier chip 2 in this example includes a recording device 30 for registration identifiable signal, indicating what Alicia of the analyte in the sample.

The second line describes an implementation option of the recording system described above for the first aspect, and this recording system is designed to include at least one of a sensor chip 1, containing the first soluble layer 5, the second biologically active surface layer 4 and at least a third of the soluble layer. Partly with reference to the drawing figure 3, the desired variant implementation of sensor chip 1 is shown for use in accordance with the present invention. A sensor chip 1 may include a carrier chip 2 with the surface 3, which includes the recording surface 33, the first soluble reagent layer 5 containing soluble matrix 7 and at least one label 6; the second layer, which represents the biologically active surface layer 4, and at least one of the third intermediate layer.

Examples of at least one of the third layer are layer 8 and the layer 9 in the role of a protective and/or calibration, and/or buffer layers. In other words, can be used the protective layers can be used in layers, auxiliary calibration and related to calibration layers, and can also be used for buffer layers. Apply multiple layers on the biosensor may be advantageous. As an example, the layer 8 may be made to the AK buffer layer, for example, as the layer containing biologically active components, in order to suppress the binding of the label 6 with capture probes 41, attached to the surface layer 4, in the manufacturing process of the biosensor. As another example, materials for calibration can be added to a sensor chip in order to provide a calibration check using the touch chip 1. As another example, you can add the covering layer 9, which acts as a protective and separable layer against impurities, such as organic pollutants, highlighted by the surrounding material of the cartridge during processing or storage. When a sensor chip 1 comes in contact with the liquid sample, soluble matrix layer usually can be dissolved, thus providing reagents, labels, and (optional) calibration reagents. Preparation of additional layers, as shown in the present invention may be capable of storing labels 6 in a soluble matrix 7 without their binding to the capture probes 41, attached to the surface layer, in the manufacturing process of the biosensor. The latter is desirable because biological molecules and/or the relationship may change during storage and processing, for example, leading to the formation of non-specific relationships between markers 6 and the surface of the sensor 3, which make biosensor is prigodnim for analysis by the method of substitution, of response inhibition, competitive analysis, or other analysis. Therefore, an advantage of certain embodiments of the present invention is that of communications label surface is actually formed directly in the process of analysis with the test liquid. In a third separate embodiment presents a sensor chip, similar to those described according to the first aspect or according to either the first or second variant of implementation, and the biosensor is a multi-biosensor, and the molecule-target is recorded by analysis type of analysis by the method of substitution, response inhibition or competitive analysis. The sample can be, for example, saliva testing, wrong application than the present invention is not limited. The surface of the sensor 3 may be secured by analogues of the drug 41 on the recording surface 33. A sensor chip 1, as a rule, contains the first soluble reagent layer 5, with label 6 can be embedded in the soluble matrix 7. Tag 6 can be provided with one or more antibodies to drugs 61. When the liquid sample is supplied to a sensor chip, matrix 7, usually dissolved in a liquid and labels 6 become mobile. Figure 4 schematically depicts the change in bulk concentrationC l,m,smoving labels 6 near the surface of the sensor 3 as a function of time. The duration and shape of the reaction peak is determined, for example, the thickness of the layer 5, the dissolution rate of the material layer 5, and the speed of movement of the labels 6 in the liquid sample.

Figure 4 arrow 150 indicates the time of wetting the recording surface, and the arrow 152 shows the point at which the labels become mobile. Becoming mobile, label 6 into the surface of the sensor 3 in very high concentrations. The time needed labels 6 to contact with the recording surface 33, is very short, due to the high short-term concentrations labels and high biological affinity labels 6 to analogues of the drug on the recording surface 33. Arrow 154 shows the point at which the label largely dissipate from the recording surface or contact her. While antibodies 61 are exposed to molecules of a drug, i.e. target molecules in solution. When drug molecules bind with antibodies 61, linking labels 6 analogs of drugs 41 on the recording surface 33 is attenuated and/or removing labels from 6 analogues of the drug on the recording surface 33. Changes over time for labels 6 associated with the recording surface is d 33, schematically depicted in Figure 5 for low, moderate and high concentrations of target molecules in solution. The sample is wetted first time, shown by the arrow 150. For this reagent layer 5 and the data labels 6 duration and shape of the surface concentration associated labelsC_l,b,sassociated with the surface of the sensor, as a function of time for different concentrations of analytes shown in figure 5. Curve 162 shows the concentration associated labels for low concentrations of analytes, showing that the concentration associated labels remains high. Curve 164 shows the concentration associated labels for higher concentrations of analytes, showing that the concentration of mobile tags a few drops after reaching a peak. Curve 166 shows the expected concentration related tags for even higher concentrations of analytes, showing that the fall of the concentration related tags even stronger after reaching the peak. When the surface concentration of the labels 6 measured by the sensor, the concentration of the target can be obtained from the time dependence of the signal and/or signal magnitude over time. Check labels 6, as a rule, can occur on the recording surface 33 by means of optical or magnetic Desk. For example, the magnetic sensor 30, such as a hall sensor, can is to be embedded in the carrier chip 2 to the registration binding magnetic label 6 with the recording surface 33. The number of registered marks 6 directly or inversely proportional to the number of target molecules and hence can be determined concentration of the target. The scan speed is limited by the speed of the Association between the drug and the antibody. For this antibody, the probability ofpwhat is the connection of the drug-antibody increases linearly with time in the limitp<1. The increase in the probability per unit of timedp/dtgiven by the equation:

dp/dt=k_on[T]

wherek_onthe constant Association for the binding of the drug to the antibody, is equal to, for example, 10⁵l/(mol×s) for the drug-antibody, [T] is the concentration of drug in the liquid, equal to, for example, 100 nmol/l givedp/dt= 0,01/S. This means that in ten seconds due to the drug-antibody formed with a probability of 10%.

In a fourth separate embodiment, presents such a sensor chip as shown in figure 2, and biosensor coated soluble layer of the reactant molecule and the target is recorded by enzymatic analysis. The reagent layer may be a thin layer as described above may also be provided with multiple layers. The sample may, for example, be a liquid sample containing the enzyme for the registration of the enzymatic activity of this enzyme. Typically, the enzyme activity can be is expressed in units defined as the amount of enzyme required to convert a certain amount of the substrate for a certain period of time. Specific activity can also be expressed in units of volume of the sample. The enzyme may be a chip off the enzyme, such as protease or nuclease, and the activity of the cleavage can be determined. In the context of the present invention, the enzymes are defined as biologically active agents that enhance cleavage, for example by using endopeptidase or endonucleases, fragmentation, for example by using ectopeptidases or economies, or modification (for example, by kinases or phosphatases, using oxidase or reductase) of the substrate with the formation of the product, i.e. the product of cleavage. The enzyme can also be modifying enzyme such as a kinase or phosphatase, which adds or removes biochemical agents, such as phosphate group. In other words, the molecule-target can also be specifically converted by the enzyme oxidase or reductase. Thus, the enzyme may, for example, be located in a thin film of dried reagent on the recording surface. After wetting the target, as a rule, subjected to the conversion and one of the reaction products is registered or to be processed further. Thus can be defined modify the sure activity. As a rule, the splitting can lead to the release of at least two parts of the product. The substrate of the enzyme may be a protein or peptide, as they can easily be converted into a product by the enzymatic reaction. Alternative substrates of enzymes may be other biological and chemical substances such as nucleic acids, lipids, carbohydrates, and chelators. The substrate of the enzyme can be part of the reagent layer 5 or may be immobilized on the sensor surface 3. The reagent layer 5, as a rule, able to dissolve in a liquid sample, the effect of which he is exposed. The reaction occurring near the surface of the sensor 3, is a ratio of specific activity of the enzyme in a liquid sample. The rate of the enzymatic conversion can be estimated as follows. For the substrate of the enzyme in the device, the probability p that the substrate is converted by the enzyme, increases linearly with time in the limitp<1. The increase in the probability per unit of timedp/dtis given in the equation:

dp/dt=k_on[E],

wherek_on- constant conversion, equal to, for example, 10⁶l/(mol×s), [E] the concentration of the enzyme in a liquid sample, is equal to, for example, 100 nmol/l, givedp/dt= 0,1/S. This means that the probability of preobrazovaniya after one second is 10%. When the concentration of the enzyme [E] in the liquid sample is, for example, 1 µmol/l,dp/dt= 1/s, which means that in 1 with the substrate of the enzyme near the recording surface is transformed with a probability of almost 100%. As described above, the rapid dissolution and diffusion of the reactants is a process, having the character of spike. When diffusion is the main transport mechanism, a suitable layer thickness ofLcan be calculated by the formulaL≈(Dt)^1/2whereD- constant diffusion of the active ingredient dissolved in the reagent, andt- the desired reaction time. When the substrate of the enzyme - small protein D is of the order of 10^-10m²/s after protein is released into the solution. When the required reaction time is 1 s, a suitable thickness is about 10 μm. Significantly more than a thin layer will give a strong time dependence of the concentration of the substrate of the enzyme in the reaction. Significantly thicker layer will absorb an excessive amount of reagent and, in addition, will create a distribution of the target molecules, for example, products of the enzymatic conversion of the substrate remote from the sensor surface 3. When the enzyme is associated with a larger object, such as with nanoparticle size 300 nm,Dis of the order of 10^-12m²/C. When the desired time is Yeni reaction with 10, a suitable thickness L is approximately 3 microns.

In the first specific example of the fourth variant of implementation, the reagent may contain a labeled substrate of the enzyme (6, 61). The surface of the sensor 3 is usually covered with the substrate-binding agent 4, for example, protivotarannymi antibodies. Such agents can be embedded in a soluble protective layer, for example, contains sugar molecules for hydration. Usually after wetting of sensor chip 1 fluid sample two events can occur in parallel. Enzymes from the sample can usually be split label 6 from tagged substrate of the enzyme (6, 61), thus freeing the substrate, while protivocesterne antibodies 41 on the surface of the sensor 3 captured and marked substrate of the enzyme (6, 61), and (split, devoid of labels) the enzyme's substrate 61. Thus, splitting activity of the investigated enzyme reduces the possibility of binding labels 6 with the surface of the sensor 3 and/or releases that almost all associated labels 6 from the surface of the sensor 3.

In the second specific example of the fourth version of the implementation of the surface of the sensor 3, as a rule, covered by the product-binding agents 41, selectively connecting the product of the enzymatic conversion.

In the third specific example of the fourth version of the implementation of the enzyme's substrate 61 in words the reagent 5 pre-associated with the mark 6 and carries a molecular tag, such as Biotin. The surface of the sensor 3 may be covered with binding to the molecular tag agent such as streptavidin.

In the fourth specific example of the fourth version of the implementation of the reagent layer 5 may contain a label 6, previously associated with the substrate-binding agent 61, such as protivocesterne antibody. The surface of the sensor 3 is covered with the enzyme's substrate 41. Mark 6 can be molecular marked label, such as Biotin. The surface of the sensor 3, as a rule, can be associated with the enzyme's substrate 41, which is supplied available to bind to the molecular tag agent such as streptavidin.

In the fifth specific example of the fourth version of the implementation of the reagent layer 5 contains the enzyme's substrate 61 with the first and second molecular markers, and also includes a label 6 with agent capable of binding the first molecular tag. The surface of the sensor 3 is connected with the capture probe 41, which may associate the second molecular tag. Suitable pairs molecular tag/binding agent can be, for example, avidin/Biotin, streptavidin/Biotin, hapten/antibody, protein/antibody, peptide/antibody, protein/carbohydrate, protein/protein, nucleic acid/nucleic acid, protein/nucleic acid, hapten/nucleic acid.

In the sixth specific example of the fourth the CSOs variant implementation of the reagent layer 5 contains a substrate of the enzyme and the product of the enzymatic conversion is logged in competitive analysis the analysis by the method of substitution - reaction inhibition, as described above.

In the seventh specific example of the fourth version of the implementation of the reagent layer 5 contains the analyte-specific enzyme and product-sensitive component that is pre-associated with the mark 6. For example, the target analyte in the sample can be glucose. After wetting the touch chip of glucose molecules in the sample are converted into hydrogen peroxide by the action of glucose oxidase, present in the reagent layer 5. This product of oxidative reactions sequentially interacts with the oxidation-sensitive agents, for example with the residue of the cysteine embedded in a protein, such as phosphatase. Immunoasays conducted with antibodies 41, sensitive to the degree of oxidation of oxidizable agent, indicates the level of glucose in a liquid sample.

In the following example, the desired analyte has regulatory effect on the enzymatic conversion and represents, for example, a promoter, activator, inhibitor or cofactor. The dry multilayer reagent includes an enzyme and its substrate. After wetting biosensor enzyme and the substrate of the enzyme dispersed in the solution. The next step is formed product of the enzyme with a speed that depends on the concentration of the analyte in solution. After which the product may be the dawn of esterban. Probably, the substrate of the enzyme is pre-associated with the mark register 6, and the recording surface 33 is supplied with antibodies to the product 41. In another embodiment, a similar product previously associated with the mark registration 6 or with the recording surface 33. Probably mark registration 6 is a magnetic particle.

In the following example, the product-binding agent 61 is pre-associated with labels 6 and similar product 41 is located on the surface of the sensor 3. In a further example similar product 61 is pre-associated with the marks 6 and the surface of the sensor 3 is covered with the product-binding agent 41. As mentioned above, also applies to structures with one or more molecular tags.

Use the following desirable implementation is in the magnetic excitation in the process of dispersion. This process is also referred to as the process of resuspendable or redispersible. In this embodiment, the dried reagent is dispersed, while the magnetic excitation is applied during and/or after redispersion. In the most preferred embodiment, the surface of the sensor by applying a magnetic field attached reagents associated with magnetic particles. It turned out that this magnetic stimulation accelerates the process of binding to the surface of the sensor.

If desired, the next step excitation, unbound or non-specific bound magnetic particles can be sequentially removed from the surface of the sensor. This magnetic excitation may need to be carried out with sensor chip; a reading system that includes a device for magnetic excitation, is located on one side of the sensor surface, and the second magnetic excitation is located on the other side of the sensor surface.

In another example, the molecule-target is a small organic molecule, usually too small to be zaregistrirovannoe using the available capture molecules such as antibodies. In this case, the reagent layer may contain enzymes and suitable agent for synthesis, such that after wetting the enzyme creates a complex substrate-target, which is easier to register, for example, using highly specific to the target-substrate complex antibodies.

According to the fifth embodiment of the present invention, the investigated analyte is a nucleotide, and the methods of the invention involve the use of at least one labeled analyte-specific probe, which represents a nucleotide probe, the sequence of which is complementary to or corresponds to at least part of the investigated analyte, in particular with ecifica to the analyte sequence. This nucleotide probe is associated with a label in order to allow specific registration of the analyte, as described above. In the recording device may also include a temperature control for the registration of such nucleotides that certain temperature conditions may be favorable for the respective reactions hybridization.

In another separate embodiment, in accordance with any of the above described variants of implementation, soluble layer on the sensor chip may include a known amount of target molecules or analogues targets for vnutrikorovogo calibration analysis. The latter allows to calibrate a sensor chip, thus allowing to obtain more accurate results.

In another separate embodiment, in accordance with any of the above described variants of implementation, soluble layer 4, 5 on the sensor chip may contain inactive substances 41, 61 and/or the inhibitor, and/or a blocking agent.

In another separate embodiment, in accordance with any of the above described variants of implementation, soluble layer on the sensor chip may include probes for capturing 41, 61, which are mutually biologically active but inactive for components normally present in the sample.

Once an individual is the version of the implementation, in accordance with any of the above described variants of implementation, soluble layer on the sensor chip may contain at least one component, the biochemical activity of which is reduced, for example, by folding, shielding, protective coating or masking, or due to the presence of a protective agent. The protective agent may, for example, be used to save a component during processing and storage. Soluble layer on the sensor chip may also include activating enzyme, for example, to cancel the effect of the protective agent. For example, when the surface is wetted, the activating enzyme disperses in the solution and activates the biologically active component.

Various embodiments of the present invention provide for a large number of convenient biotests quick and costߛeffective manner. Multiple chromogenic labels can be used in the technique of microarray, flow cytometry, Desk-based resonance energy transfer fluorescence (CHEF), which is due to interaction between excited States of the electrons of two chromogenic molecules of pigment; registration technique based on molecular beacons, such as, for example, registration of the nucleic acid to the slots in real-time or quantitative PCR in real time, in technology enhanced surface registration, such as enhanced surface Raman) Raman spectroscopy (SERS, surface-enhanced Raman spectroscopy), technology enhanced surface fluorescence (SEF, surface-enhanced fluorescence) or technology enhanced surface resonance Raman scattering (SERRS); in a microfluidic technique of registration etc. In some embodiments, the implementation of the registration system of the present invention is an epifluorescent biosensor, which means that the exciting light falls on top of a surface, but it (the system) may also be a transmission sensor, which means that the exciting light falls on the surface of the bottom and passes through the biosensor.

Further, the invention provides a method of manufacturing system in accordance with the present invention, the method includes the steps:

a) supply of sensor chip of the recording surface,

b) bringing the surface into contact with a liquid mixture containing the reagent, and the liquid mixture is a buffer mixture containing no surfactant,

c) drying the buffer mixture with the active ingredient for the formation of a layer on the recording surface.

It has been unexpectedly discovered that the use of dried buffer mixture, almost not with the holding surface-active substances, such as Tween, leads to increased specific binding component targets, such as magnetic particles with the surface of the sensor. In this context, "almost not containing surfactants" means less than 0.01%, preferably less than 0.001% surfactant, more preferably of 0.0001% surfactant.

The present invention also provides a solid phase microtechnology suitable for highly parallel of microprosodic and highly parallel processing. It provides multiplexing in diagnostic tests. The technique allows rapid selection of the parameters of the reagent, such as layer thickness, the composition of the reagent, a combination of different layers. Registration can be done by sensors embedded in the carrier chip or external devices (e.g., through visualization).

An advantage of certain embodiments of the present invention that the optimum conditions for analysis are generated instantly upon contact of the liquid sample and the soluble layer (s) of reagent that reduces the time required for the analysis and increases the sensitivity and specificity analysis. An advantage of certain embodiments of the present invention that is achieved justify the cost of registration, for example, through use of the application of thin layers of reagent and, ultimately, needs minimal amounts of reagents.

An advantage of certain embodiments of the present invention in that the sensor or the tag in the analysis process may occur neoplasm absorbing layers. The reagent may, for example, contain spectacular or the registered agent with a molecular label, for example biotinylated antibody or biotinylated molecular beacon, then as a tag or sensor contain the agent that communicates with a molecular label. The growth of biological complexes may have advantages for short-lived complexes and complexes, which tend to form non-specific relationships during long time periods (for example, prone to the formation of clusters of particles, nonspecific binding to the surface of the sensor).

An advantage of certain embodiments of the present invention in that the biosensor provided with a thin layer of reagents on the surface of the sensor. When a sensor chip is wetted with liquid reagents are quickly dissolved and the surface of the sensor exposed to the burst of the reactants, i.e. the surface of the sensor abruptly exposed to chemicals in high concentrations. Thanks to a thin layer and high speed test, the matrix of sensors can be obtained using various Rea the clients and independent tests. The use of multiple layers allows you to conduct nutricion calibration tests. The proposed biosensor has the potential for rapid tests, low cost of reagents, for multiplexing, calibration and use of small sample volume. In other words, can be achieved in a short time tests and almost instant registration label.

An advantage of certain embodiments of the present invention is the possibility of registration of small organic molecules in a magnetic biosensors. Usually small organic molecules can be too small to be registered by using the available capture molecules such as antibodies, while due to enzymatic transformations, for example, due to merger, registered formed complexes. Providing the enzyme and/or the appropriate agents to merge, in the reagent layer, it is possible to achieve a rapid and sensitive registration.

Other possible to achieve the objectives of the scheme biosensor with soluble layers of reagents carrying out the present invention will be obvious to experts in this field.

It should be understood that, although preferred, specific design and structure, as well as the materials discussed here are applied to the device, with testwuide the present invention, various changes or modifications in the form and details may be made without departing from the scope of the essence and scope of the present invention.

The invention is illustrated in the following non-limiting examples.

Example 1

As a model system was used morphine competitive analysis, in which the morphine from the liquid sample competes with morphine immobilized on the sensor surface, for binding sites on the magnetic particles connected with protivopanikovye antibodies. Frustrated total internal reflection (frustrated total internal reflection FTIR) was employed to record the magnetic particles on the surface of the sensor manufactured by the method of injection molding of cycloolefinic polymers. FTIR suitable for monitoring binding of the magnetic particles on the surface of the sensor in real time.

Buffers and reagents

MES salt buffer (25 mm MES, 150 mm NaCl, 2 mm EDTA, 0.05 % of Tween20, pH 7,4), borate buffer (50 mm sodium borate, 0.05 % of Tween20, pH 9), covering the buffer (15 mm sodium carbonate, 35 mm sodium bicarbonate, 0.05 % of sodium azide, pH 9,6 ), drying buffer (50 mm TRIS-HCl (hydrochloride), 1 %/5 % BSA (bovine serum albumin), 5 % trehalose/sucrose), buffer redispersible (50 mm TRIS-HCl (2-amino-2-hydroxymethyl-1,3-propandiol), 0,1 % BSA, 0.05 % of Tween20, pH 8) or (76 mm Na₂HPO₄, 4 mm KH₂PO₄, 400 mm NaCl, azide, 0.1 % of Triton X405, pH 8. BSA-morphine solution (10 μg/ml BSA-morphine in 15 mm Na₂CO₃, 35 mm NaHCO₃, 0,03 % NaN₃at pH 9,6) and protivopanikovye antibody 1 mg/ml were kindly provided by Cozart Bioscience (Oxfordshire, UK). Carboxyl-adembeads 300 nm and the storage buffer were purchased from ADEM electronics (Pessac, France). EDC(hydrochloride of N-3-dimethylaminopropyl-N-ethylcarbodiimide) and NHS(N-hydroxysuccinimide) were purchased from Pierce (IL, USA).

Substrate

The experiments were conducted on cycloolefin polymer (COP/Zeonex) and polystyrene (cast) substrates of transparent plastic with a refractive index of about 1,52. Aqueous solutions show a large contact angle on these substrates (>90°), which indicates the hydrophobic character of the surface, which provides a well-defined location solutions.

Reciprocal bioconnection between

300 nm Magnetic Particles Carboxyl-Adembeads (ADEM electronics) were washed twice in the same volume of MES saline buffer with a hub of magnetic particles (Dynal MPC-1, a Hub of Magnetic Particles, Dynal Biotech ASA). Particles were resuspendable in MES buffered saline in a ratio of 10 mg/ml (1% dry weight). To activate the carboxyl groups of particles were preincubator for 30 minutes at 37°C with shaking at 1000 rpm) (Termomixer Comfort, Eppendorf, USA) c 40 mg/ml EDC and 40 mg/ml NHS, mixed in the ratio 1:1 with water. The activated particles were romiti once MES saline buffer, once borate buffer and finally resuspendable in borate buffer in a ratio of 10 mg/ml Before linking, in order to avoid aggregation of the particles have been processed by the sound three times in three seconds with 40%amplitude using an ultrasonic probe (VCX 130, Sonics Vibra-cell, Sonics&Materials, Inc. USA). Protivopanikovye antibodies were added to the solution containing the particles at a ratio of 2.8 μg/ml and incubated for one night (with shaking at 1000 rpm) at 20°C, after which the processed sound three times in three seconds with 40%amplitude using an ultrasonic probe. In order to inactivate the active carboxyl groups, particles were incubated with 0.1 M glycine for 30 minutes with shaking at 1000 rpm) at 20°C. thereafter, the coated particles were washed with borate buffer 2 times, transferred to a new tube and washed for the last time in two volumes of buffer storage. The particles were stored in the storage buffer at a concentration of 10 mg/ml at 4°C.

Analysis

Floor surface.Polystyrene substrates were preincubator for one night in air-conditioned atmosphere at 4°C coated with two μl of BSA-morphine with a concentration of 10 μg/ml in covering the buffer. After incubation, the surface was air-dried after it has been washed 3 times with 100 μl PBS (phosphate-salt superdirectory).

The drying process.Magnetic particles associated with protivopanikovye antibodies were resuspendable by intensive mixing, and a certain amount was transferred into a clean tube. The magnetic particles were washed three times in drying buffer using the concentrator of the magnetic particles. Particles were resuspendable at a final concentration of 2 % in the drying buffer, 1 μl of which was placed on top of the BSA-morphine floor. Drying was performed using silikagelevye packages in a closed container overnight.

The process of redispersible.The dried reagents were redispersible by adding 13 ál buffer. After redispersion, to speed up the process of binding the magnetic particles attracted to the surface by applying a magnetic field generated by an electromagnet placed under the surface of the sensor. Unbound particles were removed from the surface using a magnet located above the surface. Processes redispersible, binding of magnetic particles to the surface and cleaning was accompanied by their visualization.

3. Results

The experiments were performed using ultrafast morphine competitive analysis with a very thin layer of dry reagents on the surface of the substrate. For this the x experiment 2%solution associated with antibody magnetic particle was dried coated with BSA-morphine polystyrene substrate.

Re-suspension of 2%of the layer of magnetic particles (2 %concentration of magnetic particles associated with protivopanikovye antibodies in 1 %BSA, 5% (Trehalose), 50 mm Tris at pH 8.5) buffer containing 0 ng/ml of morphine led to the binding of the magnetic particles with the surface of the sensor during redispersion. It was found that the magnetic excitation after redispersion led to growth in the binding. These relationships were specific, as can be seen from the difference in the number of magnetic particles retained on the surface between resuspendable, excitation and magnetic clearing in the buffer does not contain morphine as compared with resuspendable, excitation and magnetic purification buffer containing 10 ng/ml of morphine.

The results showed that antibodies associated with nanoparticles superparamagnetic, located on the surface of antigen coated, two reactive layer can be located on top of each other without interacting with each other. Besides, the principle of ultra-fast (measured in seconds) redispersible, which is legkouswaivaemye way of arranging biosleep convenient for biosensors require short-time tests.

7 shows a graph of the dose-effect dependence of the optical signal due to the binding of the magnet is s particles on the concentration of morphine.

It was found that redispersion magnetic particles in the solution containing morphine showed dose-dependent decay of the signal after the beginning of the dispersion and magnetic filtration. These results show that this system of analysis is very suitable for the location of the layer associated with antibody magnetic particle exactly on the functionalized surface of the substrate.

Example 2

As a model system was used competitive analysis of morphine, in which the morphine from the sample competes with morphine immobilized on the sensor surface for binding sites on the magnetic particles associated with protivopanikovye antibodies. FTIR was employed to record the magnetic particles to the sensor surface, molded from polystyrene.

For these experiments polystyrene substrates were covered with two μl of BSA-morphine solution (10 mg/ml BSA-morphine in 15 mm Na₂CO₃, 35 mm NaHCO₃, 0,03 % NaN₃at pH 9,6), during one night in air-conditioned atmosphere at 4°C. After incubation, the surface was rinsed three times with 1 ml of H₂O, and 1 μl of 1 %-s w/v magnetic particles associated with protivopanikovye antibody was placed on top of the BSA-morphine floor. Particles were pre-diluted 1:1 in the drying buffer (1 % BSA, 5% trehalose, 50 mm Tris at pH 8.5) in the presence or otsutstvie,1-percentage of non-ionic surfactants (Tween20). Drying the magnetic particles was carried out in dry conditions (silica gel packets) within 12 hours. the upper liquid cartridge was glued on top of the optical cartridge, and redispersible dry reagent was made by adding 13 ál buffer (Na₂HPO₄of 10.76 g/l, KH₂PO₄0.577 g/l, NaCl 23,38 g/l of 0.01 % Na-azide). The optical signal was observed within one minute using a camera based on a CCD.

Fig shows the FTIR signal during redispersible, excitation and magnetic filtration. It was found that the speed and level of redispersible 1 %aqueous layer dried particles is comparable for both drying buffers and close to 90 %. This result can be obtained by measuring the optical signal clean rough surfaces and its comparison with the region where the particles.

At the stage of attraction of the magnetic particles, dried (BT) buffer not containing Tween20 (TM), reach the surface in a larger amount compared to magnetic particles, dried (T) buffer containing Tween20 (TM), which appear on the surface at a very low concentration. It is important to note that more particles are attracted to the functionalized surface and more of the particles have a chance to bind the antigen.

1. The recording system (100) for registration at mereoni of a target molecule, moreover, the recording system (100) includes at least one of a sensor chip (1), containing on the recording surface (33) of a molecule target immobilized on the recording surface, or the capture molecule to a target molecule is immobilized on the recording surface, and at least the first soluble layer (5)containing the labeled molecule to bind to the target.

2. The recording system (100) according to claim 1, in which the labeled molecule to bind to the target is a magnetic particle.

3. The recording system (100) according to claim 1 or 2, in which the labeled molecule to bind to the target is an antibody.

4. The recording system (100) according to claim 1, comprising a detector (30) to register at least one tagged molecule to bind to the target (6).

5. The recording system (100) according to claim 1, in which the first soluble layer (5) has a thickness of between 1 μm and 50 μm.

6. The recording system (100) according to claim 1, in which a sensor chip (1), in addition, contains at least the calibration layer, in order to provide calibration reagents, and calibration layer is soluble layer intended for the calibration.

7. The recording system (100) according to claim 1, in which a sensor chip (1)further contains a protective layer (9)located above the p is at least the first soluble layer (5), and provides protection, and a protective layer (9) is soluble.

8. The recording system (100) according to claim 1, in which said system includes an excitation system of a liquid, which is provided for the reaction.

9. The recording system (100) according to claim 1, in which the system includes a magnetic excitation.

10. The recording system (100) according to claim 1, in which the recording surface is porous.

11. The method (200) registering at least one of a target molecule in a sample, comprising the steps, at which
bring the sample into contact (202) with sensor chip according to claim 1,
provide interaction (204) between at least the first soluble layer on the recording surface and the liquid sample, which allows at least one tagged molecule to bind to the target to interact with at least one molecule target and
register (206) signal reception, based on the labeled molecule to bind to the target.

12. The method according to claim 11, further containing the step of excitation of liquid in which is provided a reaction.

13. A sensor chip (1) for registration of at least one of a target molecule in a sample, containing on the recording surface (33) of a molecule target immobilized on a recording surface, and the capture molecule to a target molecule, immobilized on the recording surface, and at least the first soluble layer (5)containing the labeled molecule to bind to the target.

14. A sensor chip (1) according to item 13, in which the labeled molecule to bind to the target is a magnetic particle.

15. A sensor chip (1) according to item 13, in which the capture molecule to a target molecule is an antibody.

16. A sensor chip (1) according to item 13 or 14, in which the first soluble layer (5) has a thickness of between 1 μm and 50 μm.

17. A sensor chip (1) according to item 13, which contains at least the calibration layer, in order to provide calibration reagents, and calibration layer is soluble layer intended for the calibration.

18. A sensor chip (1) according to item 13, which contains a protective layer (9)that is located over at least the first soluble layer (5), and provides protection, and a protective layer (9) is soluble.

19. A sensor chip (1) according to item 13, in which the recording surface is porous.