MXPA00006507A - Device, method and apparatus for implementing the method, for dosing at least a particular constituent in a product sample - Google Patents

Abstract

The invention concerns a device for dosing at least a particular constituent in a product sample, comprising a receptacle (110) and a cover (120) assembled to form a closed container. The invention is characterised in that said container has a vertical axis (101), the receptacle and the cover bear coaxial cylindrical walls (111, 112, 113) defining concentric annular chambers (102, 103, 104) inside the container, the walls separating chambers each comprising an opening (11a, 112a), the cover and the container can rotate relatively to each other about the vertical axis and said openings are placed in predetermined manner so that by relative displacement of the walls, the openings are positioned in a straight line or offset to communicate or isolate said successive chambers. The invention also concerns a method for using such a device and an apparatus for implementing said method.

Description

DEVICE. METHOD AND APPARATUS FOR PUTTING THE METHOD INTO PRACTICE. TO DOSE AT LEAST ONE PARTICULAR CONSTITUENT IN A PRODUCT SAMPLE The present invention relates to a device designed for the qualitative or quantitative dosing of at least one particular component in a product sample, preferably making it possible to implement the immunological dosage, as well as to a method and equipment that are designed to implement said device. Several methods have been developed to identify, detect or quantify analytes in chemical or biological samples. These methods are based mainly on the formation of complexes by affinity reaction between the elements of a specific binding pair. These reactions, of ligand / receptor type, originate for example interactions between an antigen and a specific antibody, by hybridization between two complementary nucleic acid sequences or by a recognition phenomenon between the binding site of a protein, for example an enzyme, hormone or another biological entity, and its ligand, substrate or receptor. The formation of an affinity complex makes it possible to reveal the presence of the analyte that is subsequently sought in the sample. Possibly this analyte can be quantified if it is feasible to separate the complex forms from those that remain in free state, or measure the degree of occupancy of the analyte-specific ligands. This type of method to detect and quantify an analyte present in a sample, sometimes in smaller quantities, is of great interest for research or for analytical laboratories, especially for clinical laboratories or for biological analysis. However, for routine use, one must have the possibility to apply the methods simultaneously to a large number of samples. Moreover, it is necessary to carry out various tests in a single sample and in the same sample at the same time. Therefore, in most cases, routine manual analysis protocols involve various reactions and successive manipulation steps. These multiple tests are carried out with samples in series, in very large centers, in which several tens of thousands of samples per day can be tested. Accordingly, said multiple tests can cause some difficulties and require relatively long implementation times. Additionally, due to the successive manipulations that are required, it can give rise to the presence of errors in the results. Consequently, the problem of the automation of this type of tests immediately arises, and therefore various devices have been invented to achieve automation, or at least achieve a simplification of the successive steps indicated above.

However, these devices remain mostly relatively complex or adapt only to the detection of a particular type of analytes (cells or molecules) or otherwise only allow qualitative analyzes to be carried out. Said devices are mainly described in EP 0339277 and EP 0426729. Fundamentally, EP 339 277 describes a device for developing successive analytical reactions for dosing an analyte in a test liquid sample that involves the analytical reactions between the analyte and the reagents analytes that interact with the analyte to produce a detectable response dependent on the analyte. This device comprises a closed receptacle having a horizontal axis of rotation. This closed receptacle is delimited externally by a cylindrical wall and internally includes two concentric spoon-shaped walls defining between them a sample inlet area. Between said bucket-shaped walls and the cylindrical peripheral wall, various reaction zones are defined within which specific analytical reagents are incorporated. According to this document, the sample is introduced by an entry path into the entry chamber defined between the bucket-shaped walls and an opening towards the reaction zones. By rotating said receptacle in a reciprocating movement approximately around its horizontal axis, the liquid sample is transported by gravity within the reaction zones where it interacts with the reagents and is subsequently transported to an examination zone located in the center of the container. receptacle. Said device is specially designed to avoid any centrifugation of the product when the dosage is carried out. JP HEI-5 215 750 also describes a device for detecting and analyzing cell populations comprising a horizontal circular disc mounted rotatably about a vertical axis. This open disc is covered with antibodies. It revolves around its vertical axis in such a way that the sample introduced in its center is distributed under the action of a centrifugal force on said plate. The subsequent washing steps are carried out in the same way, by introducing the rinsing liquid in the center of the dish, the liquid is discharged towards the periphery of the dish, during the rotation of the latter, while rinsing the surface on which the component to be dosed is fixed. To recover the rinse liquid, a receptacle placed under the dish is provided. Finally, WO 94 25 159 describes a device for the qualitative and / or quantitative dosing of a particular component in a product sample, comprising a substantially circular container rotatably mounted on an axis to be driven by a central housing, and in which the test chambers are formed which extend along the radii of the container and which have a density gradient. In the central part of the container, an annular centrifugation chamber is provided which is in communication with each test chamber. This annular centrifugation chamber can be divided into two parts where one of them communicates with the other part, through a top opening. The wall delimiting the first part of the centrifugation chamber is inclined in such a way that the products are transferred from the first part to the second part spilling from the delimiting wall, through the communication hole. Similarly, the wall delimiting the second part of the centrifugation chamber shows an inclined slope in such a way that the mixture is transferred towards each test chamber spilling through the communication hole between the second part of the centrifugation chamber and said test cameras. The slope of the wall delimiting the second part of the centrifugation chamber is greater than the slope of the wall delimiting the first part, such that during centrifugation, the product passes mainly from the first part of the centrifugation chamber placed near the axis of rotation of the container, towards the second part of the centrifugation chamber before being transferred to the test chambers. The invention proposes a novel configuration of a dosing device that is simple in design and easy to use and that can be manipulated individually with a minimum number of manipulations such as to carry out the dosage, and give rise to the carrying out of the dosages in proximity to the place of removal of the sample of product containing the particular component to be dosed; said device shows an improved configuration and also makes it possible to carry out the tests in series with small amounts of samples. More particularly, according to the invention, a device is proposed for the qualitative and / or quantitative dosing of at least one particular component in a product sample by marking and fixing, said device comprises a container and a cover, which are assembled for form a closed receptacle. This device is characterized in that said closed receptacle has a vertical axis, in which the container and the cover include coaxial cylindrical walls which, while assembling the container and the cover, are placed in pairs against each other, thus delimiting at least 3 concentric annular chambers inside the receptacle, mainly from the axis, an input chamber designed to receive the sample and as convenient, give rise to the marking of the component, a camera to fix and read said marked component and a camera of discharge, wherein the coaxial cylindrical walls form separations between the successive annular chambers and each comprises at least one opening, in which the assembled cover and the container can rotate with respect to each other about a vertical axis and the openings of the cylindrical cylindrical walls of the container and the cover are placed in angular positions deter mined, in such a way that by displacement of one with respect to the other, of the cylindrical walls of each pair, the openings of each pair of walls have the ability to be placed opposite each other in inclination, to be placed in communication or isolating each other from the successive annular chambers. According to a preferred arrangement of the device according to the invention, said openings provided in the cylindrical cylindrical walls of the container and the cover are arranged in such a way that the openings of a pair of cylindrical walls are opposite each other to be placed in communication between two successive annular chambers, the openings of the other pairs of cylindrical walls are placed in inclination in such a way that the other annular chambers are insulated. According to other convenient features of the device according to the invention, there are means in the cover and in the container for placing the latter. The cover includes a nozzle on the outer face of one of these coaxial cylindrical walls that are positioned outside the other cylindrical walls to form the outer peripheral edge of the receptacle, said nozzle forming a fastener or a support for rotating the cover about the vertical axis with respect to the container. The device according to the invention comprises a central through hole, which is isolated from the immediately adjacent annular chamber and designed to be threaded on a vertical axis for the rotary pulse and to put said receptacle in rotation.

At the base of the cover or the base of the container, an inlet can be provided for the inlet chamber. According to a variant of the device according to the invention, a receptacle comprising several concentric reading and holding chambers between the input chamber and the discharge chamber can be provided. The device according to the invention shows an improved ergonomic shape. Most particularly, its receptacle shows the shape of a disk. Conveniently, the container of the device according to the invention is made of a transparent material to give rise to the reading of the marked components that have been fixed in the fixing and reading chamber through the walls of said receptacle . The cover can be opaque or it can be treated in such a way to avoid parasitic radiation, it being possible for the reading to be made with the help of a CCD camera. The device according to the present invention preferably comprises in the fixing chamber at least one receiver of the component to be dosed, said receiver has been fixed in the chamber. It should be understood that according to the present invention, the terms "receptor" and "ligand" will be used to generically designate two elements that are linked by force interactions, and that therefore can also be related to a pair of antigens / antibodies or to a pair of nucleic acid / nucleic acid complementary or otherwise to a true ligand and receptor or to other force interactions. The techniques that make it possible to fix the proteins (for example antigens or antibodies) or nucleic acids on plastic or even glass surfaces are well known to those skilled in the art, the latter interacting with technologies that are currently mainly used to fix components of this same type in standard microtiter plates that are used for example in ELISA or in some form that can be adapted to have the function of the type of polymer involved. Preferably the receiver will be fixed to the base of the container, if possible in the form of a monolayer to give rise to an easier reading. In fact, when the reading medium used is a CCD camera, the radiation will pass through the base of the container and may be modified or not modified by the presence of a marked component and will therefore recover after a Second step through the base of the container. In general, the technique involved is similar to the well known immunological "sandwich" dosing procedures, that is, the element to be detected reacts with the receptor, for example the antibody; and this same is marked by another element that recognizes it, which carries either a physical marking, ie particles or otherwise a chemical marking, for example with the help of fluorescent elements or others that can yield as fluorescents.

In this aspect, according to a convenient feature of the invention, the marking particles show a sufficient diameter, preferably greater than or equal to about 100 times the diameter of the component (s) going be dosed (s) and have optical properties that allow their detection by quantification. The expression "possesses optical properties" means that said particles have the ability to reflect all the light radiation emitted by a detection system, or part of said radiation, leading said particles. Although it is possible to mark the component before introducing it into the device, the marking element that has the ability to mark the component to be dosed will preferably be placed in the entrance chamber, for example in non-fixed dry form, this involves example, labeled antibodies that recognize one of the epitopes of the antigen to be dosed, another antibody that is fixed in the fixation and marking chamber. In the device according to the present invention, the receptor that has been set is chosen as indicated above from among the following: antibodies, antigens, complementary nucleic acid sequences, real receptors for dosage components specifically that are: antigens , antibodies, nucleic acid sequences, ligands of said receptors. For immunological dosages, preferably a labeled antigen or antibody will be used to respectively dose the complementary element and another complementary element will be fixed in the fixation and reading chamber. It is also possible to provide a multiple dosage that makes it possible to dose perhaps several antigens or several antibodies. To do this, it is sufficient that the fixing and reading chamber is divided into a variety of angular sectors on which mutually different receivers are set, each one designed to fix and read a different marked component. For this purpose, it may be particularly convenient according to the invention, that an angular sector of the receiver fixing chamber be kept away to constitute an empty sector designed to carry out a start reading of said device to zero. The reading can be carried out according to the invention, by means of a CCD camera which of course can be controlled by a computerized device that will make it possible to reconstruct the dosage of each of the elements as a function of the readings that are read. will perform in the various sectors. According to a particularly convenient feature of the invention, the CCD camera is capable of counting in a discretionary manner by emitting / receiving a light signal, the number of fixed components marked in each fixing and reading chamber, to obtain a signal of digital detection. This leads to the use of particles or microspheres to mark the component or components that are to be dosed in the sample, these particles preferably exhibit a size greater than about 100 times the size of the molecules that are subsequently searched, and which have the ability to return all or some part of the luminous radiation emitted in its direction, the reflected radiation that constitutes so many events that are captured by the CCD camera used and transmitted to a computerized tool adapted to express the detected events in terms of the absolute number in a real time. The invention also proposes a method for the qualitative and / or quantitative dosing of at least one particular component in a product sample by labeling and fixing. This method is characterized in that use is made of at least one device according to the invention, which contains specific receptors for the component to be dosed, which are fixed in each fixing and reading chamber in whose procedure, a) the product sample containing the marked component is placed in the inlet chamber isolated from other annular chambers, b) the cover is rotated with respect to the container in such a way that it puts the input chamber in communication with each fixing and reading chamber, the camera discharge is isolated from the other annular chambers, c) the device is rotated about its vertical axis in such a way that the product or product sample containing the marked component is dispersed by centrifugation in each fixing chamber and reading it is united by intense interaction of the specific receptors that have been fixed in each fixation and reading chamber, d) the cube The container is rotated with respect to the container in such a way as to place the fixing and reading chamber in communication with the discharge chamber, e) the device is rotated about its axis in such a way that the excess sample is dispersed by centrifugation. discharge chamber, f) the interior of the device is rinsed with the help of a rinse liquid circulating by centrifugation through the various annular chambers of said device while reproducing the following steps above, b), c), d) and e) in such a way that it retains in each fixing and reading chamber only the marked component joined by intense interaction to the fixed receivers, g) said marked component is detected and dosed through the wall (s) of said device. The method according to the invention can be automated and is "generic", and can be applied to the detection and quantification of target substances, whether molecular, particulate, vesicular or cellular. The invention also proposes an apparatus for implementing the aforementioned methods, characterized in that it comprises a vertical axis of rotary impulse, on which the devices according to the invention are screwed, means for keeping said devices distanced from each other, means for the bidirectional rotary pulse of said vertical pulse axis, means for receiving the samples of injection product and rinsing liquid within the input chambers of said threaded devices on the pulse axis, and means for rotating the covers of the devices in relation to the containers in such a way as to put into communication various successive annular chambers of these containers or to isolate them and a means to read the marked agents that have been fixed. Therefore, by virtue of the apparatus according to the invention, it is conveniently possible to carry out automatically, according to the method according to the invention, using the device according to said invention, the dosages of several components of a sample and even of the same sample, or of a component and even the same component in particular, in different samples of different products.

The description below together with the accompanying drawings, which are provided in the form of non-limiting examples, will clearly illustrate what the invention consists of and how it can be modalized. In the accompanying drawings: FIG. 1 is a top view of the cover of one embodiment of the device according to the invention, FIG. 2 is a sectional view along the plane AA of FIG. 1, FIG. 3 is a top view of the container of the embodiment of figure 1 of the device according to the invention, - figure 4 is a sectional view along plane AA of figure 3, - figure 5 presents two curves of detection for a given component in a serum, by fluorescence and by quantification of labeling microspheres according to the invention, and - Figure 6 presents two detection curves for a given component in a serum, by means of the ELISA method and by quantification of microspheres according to the invention. Represented in figures 1 to 4 as separate pieces, there is a device for the qualitative and / or quantitative dosing of at least one particular component in a product sample by marking and fixing.

Said device is conveniently used to carry out the immunological dosing, the detection of microorganisms, the dosing of contaminants or otherwise to reveal a particular nucleic acid sequence. Said device comprises a container 110 and a cover 120 that are designed to be assembled and form a closed receptacle. As shown in the figures, the container 110 and the cover 120 show a general circular shape, with central axis of symmetry 101, so that when they are assembled, said closed receptacle thus formed shows the shape of a disc with a vertical axis 101 The container 110 and the cover 120 each show a base 110a, 120a including coaxial cylindrical walls 11, 112, 113; 121, 122, 123 (in this case, they are three in number). While said container 110 and said cover 120 are assembled, these coaxial cylindrical walls are placed by themselves in pairs against each other thus demarcating three concentric annular chambers 102, 103, 104 within the closed receptacle. Starting from the axis and proceeding towards the outside, said cylindrical walls mainly delimit an inlet chamber 110 designed to receive the sample of the product and as convenient, give rise to the marking of the component to be dosed, for example an antibody marked with a particle in dry form, a chamber for fixing and for reading said marked component 103, comprising, for example, an antibody that was fixed to the base of the container, and a discharge chamber 104. According to the embodiment shown , the cover exhibits a diameter slightly larger than the diameter of the container, so that the cover is placed by itself in the container. The cylindrical wall 123 of the cover 120, whose wall is located outside the other coaxial cylindrical walls 121, 122, then forms, according to the embodiment shown, the outer peripheral edge of said receptacle. The coaxial cylindrical walls 111, 112, 113 of the container 110 are designed to stand on their own against the inner walls of the cylindrical cylindrical walls 121, 122, 123 of the cover 120. The cylindrical cylindrical interior walls 111, 112 and 121, 112 of the container 110 and the cover 120 forming separations between the successive annular chambers 102, 103, 104, each comprising at least one opening 111a, 112a and 121a, 112a. In this site, each of these coaxial cylindrical walls 111, 112; 121, 122 comprises three openings 111a, 111b, 111c, 112a, 112b, 112c; 121a, 121c; 122a, 122b, 122c regularly distributed over the profile of each wall while they are placed in pairs around 120 degrees. In the exemplary embodiment shown, the openings of the coaxial cylindrical walls of the container 110 and the cover are formed by notches.

Said apertures of the coaxial cylindrical walls of the container 1 10 and of the cover 120 are placed in certain angular positions, and the assembled cover 120 and the container 1 10 have the ability to rotate with respect to each other on a vertical axis 101 of such so that by displacement of one with respect to the other of the cylindrical walls of each pair forming a separation of successive annular chambers, said openings of each pair of the walls have the ability to be placed opposite each other or in inclination, to communicate or isolate said successive ring chambers together. According to the typical case, the openings 1 11a, 1 1 1 b, 1 1 1c created in the cylindrical wall 11 1 of the container 1 10 are respectively placed facing the openings 1 12a, 1 12b, 1 12c created in the wall cylindrical 1 12 of the container 1 10. Indicated in another way, each opening 111a, 111 b, 111c created in the internal cylindrical wall 111 of the container 110 is placed facing the corresponding opening 12a, 1 12b, 1 12c created in the wall cylindrical coaxial 112, placed outside said wall 1 1 1. On the other hand, the openings 121 a, 121 b, 121 c formed in the cylindrical wall 121 of the cover 120 are placed in an irregular manner with respect to the openings 122a, 122b , 122c created in the cylindrical coaxial wall 122 of the cover 120, whose wall is positioned outside said cylindrical wall 121 with an inclination of about 60 degrees, such that the openings created in a wall are not placed facing the openings formed in another successive coaxial wall of the cover.

Accordingly, with said arrangement, when the cover 120 is assembled with the container 110, it is positioned with respect to the latter in such a way that the openings of a pair of cylindrical walls face each other to place two successive annular chambers in each case. communication, the openings of the other pair of cylindrical walls are placed in inclination so that the other successive annular chambers are insulated from each other. Additionally, as shown in Figures 1 to 4, the cover 120 and the container 110 are provided with graduated positioning means. According to the embodiment shown, said graduated positioning means of the cover 120 and of the container 110 comprise, on the one hand, an opening 123a that extends over an angular sector of the cylindrical wall 123 of the cover 120 that is placed outside of the others. cylindrical cylindrical walls and form the outer peripheral edge of the receptacle, and on the other hand, a radially extending nozzle 113a while projecting from the cylindrical wall 113 of the container 110 designed to be positioned against the outer cylindrical wall 123 of the cover 120 said nozzle 113a can be coupled to said opening 123a of the outer wall 123 of the cover 120 and to navigate in this opening 123a during the relative rotation of the cover 120 and of the container 110, in such a way that it acts as an adjoining between the two end side edges 123'a, 123"a of this opening 123a.

At this site, the opening 123a created in the outer wall 123 of the cover 120 is modeled by a notch extending around 70 degrees. The two adjacent positions of the nozzle 113a in the opening 123a correspond to two determined relative positions of the cover 120 and the container 110. The first adjacent position of the nozzle 113a against the end edge 123'a of said opening 123a, corresponds to the communication of the input chamber for the product sample 102 with the camera for fixing and reading the marked component 103, and the discharge chamber 104 is isolated from the other chambers. The second adjoining position of the nozzle 113a against the other edge end 123"a of said opening 123, here corresponds to the communication of the fixing and reading chamber 103 with the discharge chamber 104 and with the isolation of the inlet chamber 110 of the other chambers To facilitate relative rotation of the cover 120 and the container 110, the cover 120 comprises a radially extending nozzle 123b while projecting from the outer face of the cylindrical wall 123 positioned outside the other cylindrical walls, said nozzle 123b forms a fastener or a support for rotating the cover 120 around the axis vertical 101 with respect to container 110 Container 110 and cover 120 each include a central through hole 105 insulated from the immediate adjacent annular chamber, here, inlet chamber 102, by a coaxial cylindrical wall 105a, 105b. When the cover is assembled with the container, the walls 105a and 105b of the container 110 and the cover 120 will be placed by themselves against each other and the closed receptacle thus formed comprises a central through hole, insulated by the two cylindrical walls 105a, 105b positioned against each other, from the immediately adjacent annular chamber 102. This central passage hole 105 is designed to be threaded about a vertical axis for the rotary pulse and to set said receptacle in rotation. The hole 105 here shows a diameter of about 4 mm. The openings created in the coaxial cylindrical walls of the container 110 and of the cover 105 to put the annular chambers of the receptacle in communication, show a width of about 5 mm. As shown very particularly in FIG. 1, at its base 120a, the cover comprises a hole 102a that arises inside the entry chamber 102. It is positioned adjacent to the passage hole 105 since the entrance chamber is immediately adjacent to the entrance chamber. said through hole105.

Of course, this entry hole could be provided to the entry chamber according to the variant (not shown) in the base 110a of the container 110. The container 110 is made of a transparent material in such a way as to allow the reading of the marked components that have been fixed in each fixing and reading chamber, through the lower wall of said container, for example with the help of a CCD camera, by radiation transmission and reflection. The cover can then be opaque, that is, treated to avoid spurious radiation. Conveniently, the container and cover are manufactured by molding a plastic, and the coaxial cylindrical walls are formed together with said cover. Any plastic material that is conventionally used to "coat" molecules can be used to modalize the device according to the invention. For example, use will be made of polystyrene, or preferably, a plastic ZYLAR (registered trademark) said plastic shows a very high fixing capacity in terms of "coating". According to a variant of said dosing device, it can be conveniently considered, for the fixing and reading chamber 103, to be divided into a variety of angular sectors in which mutually different receivers are set, each designed for the fixing and for reading a different marked component. According to this variant, it may be particularly convenient to provide an angular sector of the fixing chamber that is separated from the fixed receivers and thus constitute an empty sector on which it would subsequently be possible to carry out the reading and determine the zero start of the device. According to another variant, a receptacle can also be provided which comprises between the input chamber and the discharge chamber a plurality of concentric successive fixing and reading chambers for fixing different marked components. The dosing device constructed by assembling the container 110 and the cover 120 in such a manner as shown in FIGS. 1 and 3, allows the initiation of a procedure for the qualitative and / or quantitative dosing of at least one particular component in a sample of product by fixing and marking. This procedure will be described below. According to this method, use is made of at least one device of the type described above with the two elements, the container and the cover, as shown in figures 1 and 3, assembled to form the closed receptacle, which contains receivers specific for the component to be dosed, and that are fixed in the fixing and reading chamber. During a first step a), the product sample containing the labeled component to be dosed is placed in the inlet chamber insulated from the other annular chambers of said receptacle.

Subsequently, during step b), the cover is rotated with respect to the container in such a way that it places the input chamber in communication with the fixing and reading chamber, and the discharge chamber is isolated from the other annular chambers. Therefore, during step c), the device is rotated on its vertical axis in such a way that it is dispersed by centrifugation in the fixing chamber and reading the product sample containing the marked component to be dosed, the latter then performs a binding by intense interaction with specific fixed receivers in the fixation and reading chamber. It should be noted that the rotation of the device allows the transfer of the product sample from the entry chamber to the fixation and reading chamber, and also the agitation of the sample inside this chamber to cause the marked component to be linked with the receivers that were fixed. During a next step d), the cover is rotated with respect to the container in such a way that it places the fixing and reading chamber in communication with the discharge chamber, and then, during a step e), the device is rotated on its axis in such a way that it disperses by centrifugation the excess sample inside the discharge chamber. It should be noted that for this purpose, as shown in Figures 3 and 4, a circular rod 114 is provided in the base 110a of the container 110 of the device in proximity to the inner cylindrical wall 112 that forms the separation between the chamber fixing and reading, and the discharge chamber, this circular rod 114 forms an edge that has no return with respect to the surplus sample discharged by centrifugation in said discharge chamber, or otherwise with respect to the rinse liquid recovered in this camera as will be described below. During step f), the interior of the device is rinsed several times with the help of a rinse liquid which is circulated by centrifugation through the various annular chambers of said device while reproducing the following steps b), c), d) and e) to eliminate the other components of said product that may be adhered to the internal walls of the device or otherwise bonded by weak interaction (such as adsorption) to specific receptors that are fixed in the fixing chamber and reading. Therefore, only the marked component, bound by intense interaction to the fixed receivers in said chamber is retained in the fixing and reading chamber after rinsing. It is then possible in step f) to detect and dose through the wall (s) of said device the marked component attached to the fixed receivers in such a way as to perform the qualitative and / or quantitative dosing of this marked component. This detection can be carried out conveniently according to the method according to the invention, with the aid of a CCD camera. To do this, the marking of the component to be dosed must be carried out physically or chemically with the aid, for example, of fluorescent microspheres or those that have been produced as fluorescent. The reading of the marked agents that have been fixed, can be carried out along the radii of the fixing and reading chamber. In a very particular way, according to the method according to the invention, with the help of a CCD camera, the number of marked components that have been fixed in each fixing and reading chamber is quantified. This is possible by using, as marking elements that can mark the component to be dosed, particles or microspheres preferably about 100 times larger than the molecules that are subsequently searched, and conjugated with the antibody or developer antigen. For a better resolution of the CCD camera, particles whose diameter is 2 μm are preferred. These microspheres are in such a way that they reflect all the radiation or some part of the radiation they receive. They may consist of latex or any other material that allows detection and quantification. Therefore, the CCD camera captures a certain number of events corresponding to an absolute number of fixed components that are to be dosed. The CCD camera is linked to software that emits a digital detection signal. Said vision-based system then provides a real-time count of about a few units at 100,000 microspheres per mm2, with subtraction of background noise with the aid of a reference surface and appropriate dialgorithms. It also has a discriminatory energy until it can recognize and discard heterogeneous images prone to falsifying data analysis. Figures 5 and 6 show curves of results obtained with the aforementioned method of detection by quantification, according to the invention and very conventional detection methods based on fluorescence or ELISA type, for a given component that is to be dosed in a serum determined. The curves of figures 5 and 6 show on the one hand that in fact there is a correlation between the quantification of the microspheres and the concentration of the component to be dosed, and on the other hand, that the method of quantification of microspheres according to the invention is more accurate than the conventional detection methods already mentioned. In particular, the dosage by quantification of microspheres offers a dynamic scale of more than 4Log against 2Log for the ELISA method. This is particularly convenient since it considerably increases the detection limit. The results obtained with concentrated dilutions of the sample then show that the method shows a 2Log sensitivity higher than that of ELISA. The results obtained in reduced dilutions of the sample also show that the method can make it possible to perform dosages in analyte concentrations corresponding to the saturation threshold in ELISA. This can be conveniently possible to avoid or reduce any dilution effect on certain components of the sample to be tested. According to the method mentioned above, the marking of each particular component of the sample can be carried out outside, before introducing said sample into said chamber of entry of the device. As a variant, the marking of each particular component to be dosed from the product sample can be carried out directly in the entry and marking chamber, in a first step introducing a specific labeled receptor for each component that is going to be dosed, in non-fixed dry form, then in a second step introducing the product sample into said isolated input chamber so that the labeled receptor is bound by intense interaction to the corresponding component that is contained in said product sample. Of course, this method described with the aid of a device and as shown in figures 1 to 4, can be carried out with the aid of a variety of devices of this type, to simultaneously dose a component or the same component particular in a variety of different samples or to dose several particular components in a product sample and even in the same product sample.

For this purpose, an apparatus for practicing this method using a variety of devices of the type shown in FIGS. 1 to 4, which comprises a vertical axis for rotary pulse or has been conveniently provided in accordance with the invention, is provided. which is threaded to said dosing devices, means for maintaining said devices distanced from the others, and means for bidirectional rotating pulse of said vertical pulse axis to carry out the rotating centrifugation of said devices, means for injecting the product samples. and the rinse liquid within the input chambers of the threaded devices on the pulse axis, and means for rotating the covers of the devices relative to the containers, in such a way as to put the various successive annular chambers of said devices in communication or to isolate them, and means to read the fixed agents marked. The invention is in no way limited to the embodiments described and depicted, however, those skilled in the art will know how to vary it in any way in accordance with the spirit thereof.

Claims (33)

NOVELTY OF THE INVENTION CLAIMS

1. - A device for the qualitative and / or quantitative dosing of at least one particular component in a product sample by marking and fixing, device comprising a container (110) and a cover (120) that are assembled to form a closed receptacle, further characterized in that said closed receptacle has a vertical axis (101), wherein the container (110) and the cover (120) include coaxial cylindrical walls (111, 112, 113; 121, 122, 123) which, while the The container and the cover are being assembled, they are placed in pairs against each other thus delimiting at least three concentric annular chambers (102, 103, 104) inside the receptacle, mainly the shaft an inlet chamber (102) designed to receive the sample and as it is convenient to originate the marking of the component, a camera for fixing and reading said marked component and a discharge chamber (104), in which the cylindrical cylindrical walls es (111, 112; 121, 122) form separations between the successive annular chambers (102, 103, 104) each one comprises at least one opening (111a, 112a; 121a, 122a) and in that the assembled cover (120) and the container (110) have the ability to rotate with respect to one another on a vertical axis (101) and the openings of the coaxial cylindrical walls of the container (110) and of the cover (120) are placed at certain angular positions, such that by displacement of the cylindrical walls (111, 112; 121, 122) of each pair from each other, the openings of each pair of walls have the ability to be placed opposite one another at an inclination, to put into communication or isolate the other chamber from the successive annular chambers.

2. The device according to claim 1, further characterized in that said openings provided in the cylindrical cylindrical walls of the container (110) and the cover (120) are positioned in such a way that when the openings of a pair of cylindrical walls they are opposed to each other to bring into communication two successive annular chambers, the openings of the other pairs of cylindrical walls are placed in inclination in such a way that the other annular chambers are insulated.

3. The device according to any of claims 1 and 2, further characterized in that in the cover (120) and the container (110) includes a means to position the latter in a graduated manner.

4. The device according to claim 3, further characterized in that said means for positioning the cover (120) and the container (110) in a graduated manner comprises, on the one hand, an opening (123a) that extends over an angular sector of the cylindrical wall (123) of the cover (120) that is placed on the outside and forms the outer peripheral edge of the receptacle and on the other hand a nozzle (113a) that extends radially while projecting from that cylindrical wall (113). ) of the container (110) that is placed against said outer cylindrical wall (123) of the cover (120), said nozzle (113a) is designed to be coupled in said opening (123a) of the external cylindrical wall (123a) of the cover and to navigate in this opening during the relative rotation of the container cover, so that it abuts between the two end side edges (123'a, 123"a) of this opening (123a), the two adjacent positions of the boqui lla (113a) in the opening (123a) correspond to two determined relative positions of the cover (120) and the container (110).

5. The device according to claim 4, further characterized in that a first adjoining position of the nozzle (113a) in said opening (123a) corresponds to putting said sample input chamber (102) in communication with the camera to fix and reading the marked component (103), the discharge chamber is isolated from the other chambers (104), and a second adjacent position of the nozzle (113a) in said opening (123a) which corresponds to bringing the fixing and reading chamber (103) into communication with the discharge chamber (104) and to isolate the inlet chamber (102). ).

6. The device according to any of the preceding claims, further characterized in that the cover (120) comprises a nozzle (123b) on the external face of one of its coaxial cylindrical walls (123) that is located outside the other walls cylindrical to form the outer peripheral edge of the receptacle, said nozzle (123b) forms a fastener or support for rotating the cover (120) on the vertical axis (101) with respect to the container (110).

7. The device according to any of the preceding claims, further characterized in that the receptacle comprises a central passage hole (105) isolated from the immediately adjacent annular chamber (102) and designed to be threaded on a vertical axis for the impulse. rotating and set said receptacle in rotation.

8. The device according to any of the preceding claims, further characterized in that the base (120a) of the cover (120) or the base (110a) of the container (110), comprises an entrance hole (102a) towards the entrance chamber (102).

9. The device according to any of claims 1 to 8, further characterized in that between the input chamber and the discharge chamber, the receptacle comprises several concentric cameras for fixing and reading.

10. The device according to any of the preceding claims, further characterized in that said coaxial cylindrical walls (111, 112).; 121, 122) form gaps between the successive annular chambers (102, 103, 104), each comprising three openings (111a, 111b, 111c, 112a, 112b, 112c; 121a, 121b, 121c, 122a, 122b, 122c) regularly distributed over its circumference.

11. - The device according to any of the preceding claims, further characterized in that the receptacle exhibits the general shape of a disk.

12. The device according to any of the preceding claims, further characterized in that the container (110) is made of a transparent material in such a way as to allow the reading of the fixed components marked in the fixing and reading chamber through the the walls of said receptacle.

13. The device according to any of claims 1 to 12, further characterized in that it is made of plastic.

14. The device according to one of claims 1 to 13, further characterized in that the fixing chamber (103) comprises at least one receiver of the component to be dosed, said receiver being fixed on at least one of the walls of the chamber.

15. The device according to one of claims 1 to 14, further characterized in that said receiver has been fixed on the base wall (110a) of the container (110).

16. The device according to one of claims 1 to 15, further characterized in that the input chamber (102) comprises a marking element that has the ability to mark the component to be dosed.

17. - The device according to one of claims 1 to 16, further characterized in that the receptor that has been set is chosen from the following: antibodies, antigens, complementary nucleic acid sequences, real receptors for respectively dosing components that are: antigens, antibodies and sequences of nucleic acids, ligands of the receptors.

18. The device according to any of claims 1 to 17, further characterized in that the fixing and reading chamber (103) is divided into a variety of angular sectors in which different receivers each set to fix and Read a different marked component.

19. The device according to claim 18, further characterized in that an angular sector of the fixing chamber is kept separate from the receivers to constitute an empty sector designed to carry out an initial reading of said device.

20. The device according to any of claims 16 and 17, further characterized in that the marking is carried out by physical or chemical means.

21. The device according to claim 20, further characterized in that the marking is carried out with the help of fluorescent particles and / or elements or those that can fluoresce.

22. - The device according to claim 21, further characterized in that the particles exhibit a sufficient diameter, greater than or equal to about 100 times the diameter of the component (s) to be dosed and have optical properties that allow its detection by quantification.

23. The device according to claim 21, further characterized in that the particles have a diameter of 2 μm.

24. The device according to one of claims 16 to 23, further characterized in that the labeling element consists of a labeled antibody or a labeled antigen, or a labeled nucleic acid sequence or any receptor element that can be labeled.

25. The device according to any of the preceding claims, further characterized in that the cover is treated in such a way as to avoid parasitic radiation and the reading is carried out with the aid of a CCD camera. 26.- The device according to claim 25, further characterized in that the CCD camera has the ability to counting on a discretionary basis by emitting / receiving a light signal, the number of marked components that have been fixed in each fixing chamber and reading, to obtain a digital detection signal. 27. Procedure for the qualitative and / or quantitative dosing of at least one particular component in a product sample by marking and fixing, further characterized in that use is made of at least one device according to any of claims 1 to 26 which contains specific receptors for the component to be dosed, which have been fixed in each fixing and reading chamber and in which, the procedure a) the sample of product containing the marked component is placed in the isolated input chamber of the other annular chambers, b) the cover is rotated with respect to the container in such a way as to put the input chamber in communication with the fixing and reading chamber, the discharge chamber is isolated from the other annular chambers, c ) the device is rotated on its vertical axis in such a way that it disperses by centrifugation in each fixing and reading chamber, the product sample containing e the marked component, the latter is linked by intense interaction to the specific receivers that have been fixed in each fixing and reading chamber, d) the cover is rotated with respect to the container in such a way that it communicates with the reading chamber and fixing with the discharge chamber, e) the device is rotated around its axis in such a way that it disperses by centrifugation the excess sample inside the discharge chamber, f) the interior of the device is rinsed with the help of a liquid of rinse that is circulated by centrifugation through the various annular chambers of said device at the moment in which the previous steps b), c), d) and e) are reproduced in such a way that it is retained in the fixing and reading chamber only the labeled component bound by intense interaction to the fixed receptors, g) said marked component is detected and dosed through the walls of said device with the means s appropriate. 28. The method according to claim 27, further characterized in that the marking of each particular component to be dosed from the product sample is carried out before introducing the latter into the input chamber of the device. 29. The method according to claim 27, further characterized in that the marking of each component that is to be dosed from the product sample is carried out in the entry and marking chamber, introducing previously into the sample of product a specific marking element for each component to be dosed, preferably in non-fixed dry form, then introducing the product sample into said isolated input chamber so that the marking element can be linked by intense interaction to the corresponding component in said product sample. 30.- The method according to one of claims 27 to 29, further characterized in that the reading operation allows the detection of the marked component that has been fixed in the fixing and reading chamber and is carried out with the help of a CCD camera. 31.- The method according to claim 30, further characterized in that the CCD camera carries out a discretional quantification by emission / reception of a light signal of the marked components that have been fixed in each fixing and reading chamber, for obtain a digital detection signal. 32. The method according to one of claims 27 to 31, further characterized in that the reading of the marked agents that have been fixed is carried out in accordance with the radii of the fixing and reading chamber. 33.- An apparatus for implementing the method according to claims 27 to 32, further characterized in that it comprises a vertical axis for the rotary pulse on which devices according to one of the claims 1 to 26 are screwed., means for maintaining said devices spaced apart from the others, means for the bi-directional rotary pulse of said vertical pulse axis, means for injecting product samples and the rinsing liquid into the inlet chambers of said threaded devices on the pulse axis , and means for rotating the covers of the devices in relation to the containers, in such a way as to put the various successive annular chambers of these containers in communication or isolate them and means to read the marked agents that have been fixed.