EP3524982B1 - Rotatable test element - Google Patents

Description

The invention relates to a test element that is essentially disk-shaped and flat and can be rotated about a preferably central axis that is perpendicular to the disk-shaped test element plane, containing a sample application opening for application of a liquid sample, a capillary-active zone, which is a porous, absorbent matrix , and a sample channel that extends from the sample application opening to the capillary-active zone. Furthermore, the invention relates to a method for determining an analyte using the test element.

In principle, the systems used to analyze liquid sample materials or sample materials that can be converted into liquid form can be divided into two classes: on the one hand there are analysis systems that work exclusively with so-called wet reagents, on the other hand there are systems that work with so-called dry reagents. Especially in medical diagnostics, but also in environmental and process analysis, the former systems have prevailed in the field of permanently equipped laboratories, while the latter systems are mainly used in the field of "on-site" analysis.

In the field of medical diagnostics, analysis systems with dry reagents are used, in particular in the form of so-called test carriers, e.g. B. offered test strips. Prominent examples of this are test strips for determining blood sugar levels or test strips for urine tests. Such test carriers usually integrate several functions (e.g. the storage of reagents in dried form or - albeit less frequently - in solution; the separation of unwanted sample components, in particular red blood cells from whole blood; in immunoassays, the so-called bound Free separation; the dosing of sample volumes; the transport of sample liquid from outside a device into a device; the control of the chronological sequence of individual reaction steps; etc.). The function of sample transport is often accomplished by means of absorbent materials (e.g. paper or fleece), by means of capillary channels or by using external driving forces (e.g. pressure; suction) or by means of centrifugal force. Disk-shaped test carriers, so-called LabDiscs or optical BioDiscs, continue the idea of controlled sample transport using centrifugal force (centrifugal force). Such disk-shaped, CompactDisc-like test carriers enable miniaturization through the use of microfluidic structures and at the same time the parallelization of processes through repeated application of identical structures for the parallel processing of similar analyzes from one sample or identical analyzes from different samples. Especially in the area of optical BioDiscs, the integration of optically stored digital data for the identification of the test carrier or the control of the analysis systems on the optical BioDiscs is possible.

In addition to the miniaturization and parallelization of analyzes and the integration of digital data on optical discs, BioDiscs generally have the advantage that they can be manufactured using established manufacturing processes and measured using established evaluation technology. In the case of the chemical and biochemical components of such optical BioDiscs, it is usually possible to fall back on known chemical and biochemical components. The disadvantage of the optical LabDiscs or Biodiscs based purely on centrifugal and capillary forces is that it is difficult to immobilize reagents and the accuracy of detection suffers. Especially in detection systems based on specific binding reactions such. B. Immunoassays, the volume component is missing compared to conventional test strip systems, especially in the so-called. Bound-free separation.

For this reason, there have recently been attempts to establish hybrids of conventional test strips and BioDiscs, especially in the field of immunoassays. The result are BioDiscs with channels and channel-like structures for liquid transport on the one hand and bulky absorbent materials in these structures (at least in part) on the other.

WO 2005/001429 (Phan et al. ) describes optical bio-disks that have pieces of membrane as reagent carriers in parts of the channel system. The reagents are dissolved by a liquid fed to the disk, resulting in buffered reagent solutions which are then contacted with the sample.

Out of WO 2005/009581 (Randall et al. ) optical bio-disks are known which contain absorbent membranes or papers for moving a sample liquid, for separating particulate sample components, for carrying reagents and for analyzing the sample. The sample is first applied to a blood separation membrane near the outer edge of the bio-disk and migrates radially therethrough to a reagent paper located closer to the center of the bio-disk. The sample is then moved radially outwards again, ie away from the center of the Bio-Disk, and flows through a so-called analysis membrane. The movement outwards takes place via chromatography, which is supported by the rotation of the Bio-Disk and the centrifugal force that acts on the sample as a result.

US 2002/0076354 A1 (Cohen ) discloses optical bio-disks which, in addition to a channel system for transporting a liquid sample, have what is known as a “capture layer”. The latter can consist of nitrocellulose, for example. The "interceptor layer" is flown through with the help of centrifugal forces when the disk rotates.

US 2005/0014249 (Staimer et al. ) and US 2005/0037484 (Staimer et al. ) describe optical bio-disks with porous materials integrated into channels that act as chromatographic separation media. The sample liquid is forced outwards through the separating media from a sample application point near the center by means of centrifugal force and then flows radially inwards again in a channel after passing through a filter.

US 2004/0265171 (Pugia et al. ) describes a test element with liquid channels in which the sample liquid is transported by means of an interplay of capillary force and centrifugal force. A nitrocellulose strip can be provided within a liquid channel, which carries an agglutination reagent which reacts with the analyte and can thus lead to the formation of so-called bands which can finally be measured optically and thus serve to determine an analyte concentration in the sample. With the help of the nitrocellulose strip it is possible to transport the sample liquid both parallel to the centrifugal force and in the opposite direction to the centrifugal force, especially when another absorbent material, for example an absorbent nitrocellulose paper, is used to support the suction effect.

WO 99/58245 (Larsson et al. ) describes microfluidic test elements in which the movement of liquids through different surfaces with different surface properties, such as e.g. B. different hydrophilicity is controlled.

US 5,242,606 (Braynin et al. ) discloses circular, disk-like rotors for centrifuges that have channels and chambers for transporting sample liquids.

US 2005/084422 (Kido et al. ) describes test elements with microfluidic structures, which have, among other things, chambers for introducing the sample and chambers for introducing a dilution buffer, which are each microfluidically connected to mixing chambers, from which another channel branches off, which is connected to an analysis chamber.

US 5,160,702 (Kopf-Sill et al. ) discloses microfluidic structures that describe a sample chamber and a dilution chamber as separate substructures that open into a common mixing chamber. A channel leads from this mixing chamber to the fluidic areas in which the detection takes place.

A disadvantage of the concepts of the prior art is that just for specific binding assays such. B. Immunoassays a targeted control of the reaction and residence times of the sample liquid after receiving the reagents and after flowing into the porous, absorbent matrix is not possible.

The object of the invention is to eliminate the disadvantages of the prior art.

This object is achieved by the subject matter of the invention.

The subject matter of the invention is a test element according to claim 1, a system according to claim 11 and a method according to claim 7. Advantageous configurations and preferred embodiments of the invention are the subject matter of the dependent patent claims.

The test element according to the invention is essentially disc-shaped and flat. It is rotatable about a preferably central axis that is perpendicular to the disk-shaped test element plane within the test element. Typically, the test element is a circular disc, comparable to a compact disc. However, the invention is not limited to this shape of the disk, but can also be used with non-symmetrical or non-circular disks.

As a component, the test element initially contains a sample application opening into which a liquid sample can be pipetted or introduced in some other way. The sample application port is near-axis (ie, close to the center of the disk). The sample application opening can open directly into a sample channel. However, it is also possible for the sample application opening to initially open into a reservoir located behind it, into which the sample flows before it continues to flow into the sample channel. Appropriate dimensioning can ensure that the sample flows from the sample application opening into the downstream fluidic structures without further action. This can be done by hydrophilizing the surfaces of the fluidic structures be necessary and/or the use of structures that promote the development of capillary forces. However, it is also possible to allow the fluidic structures of the test element according to the invention to be filled from the sample application opening only after the action of an external force, preferably a centrifugal force.

Furthermore, the test element contains a capillary-active zone in the form of a porous, absorbent matrix that absorbs at least part of the liquid sample. The capillary-active zone has a first end remote from the axis and a second end close to the axis.

The test element also has a sample channel that extends from the sample application opening to the first end of the capillary-active zone, which is a porous, absorbent matrix and is remote from the axis. The sample channel runs at least once through a region close to the axis which is closer to the preferably central axis than the first end of the capillary-active zone which is remote from the axis.

The essential feature of the test element of the present invention is that the capillary-active zone, which is a porous, absorbent matrix, has a second end close to the axis. The first end of the capillary-active zone that is remote from the axis is in contact with the sample channel in which the sample can be moved by capillary forces and/or centrifugal forces and/or other external forces such as positive or negative pressure. As soon as the liquid sample - possibly after the absorption of reagents and/or dilution media and/or the expiry of preliminary reactions - reaches the first end of the capillary-active zone remote from the axis, it is absorbed into it and, by the capillary forces (which in the case of a porous absorbent Matrix can also be referred to as suction forces) transported through it.

The capillary-active zone is a porous, absorbent matrix, in particular a paper, a membrane or a fleece.

The capillary-active zone, which is a porous, bibulous matrix, contains one or more zones with immobilized reagents.

In the capillary-active zone, which is a porous, absorbent matrix, are specific binding reagents, for example specific binding partners such as antigens, antibodies, (poly)haptens, streptavidin, polystreptavidin, ligands, receptors, nucleic acid strands ("capture probes") and the like , immobilized. They serve to selectively capture the analyte or species derived from the analyte and related thereto from the sample flowing through the capillary-active zone. These binding partners are immobilized in the form of lines, dots, patterns in or on the material of the capillary-active zone. For example, in the case of immunoassays, an antibody against the analyte can be immobilized on the surface of the capillary-active zone, which is a porous, absorbent matrix, which then capture the analyte (in this case an antigen or hapten) from the sample and also immobilize it in the capillary-active zone. The analyte can be made detectable by further reactions, for example by further bringing it into contact with a labeled partner capable of binding, for example by a visually, optically or fluorescence-optically detectable label.

In a preferred embodiment of the test element according to the invention, the capillary-active zone, which is a porous, absorbent matrix, adjoins another absorbent material or an absorbent structure with the second end near the axis, so that this or this liquid can be absorbed from the zone. Typically, the porous bibulous matrix and the other material overlap slightly for this purpose. The additional material or the additional absorbent structure serves on the one hand to support the suction effect of the capillary-active zone, which is a porous absorbent matrix, and on the other hand as a receiving zone for liquid that has already passed through the capillary-active zone. The further material can consist of the same or different materials as the matrix. For example, the matrix can be a membrane and the other absorbent material can be a fleece or paper. Other combinations are of course also possible.

In a preferred embodiment, the test element according to the invention is characterized in that the sample channel has zones of different dimensions and/or for contains different functions. For example, the sample channel can contain a zone containing sample-soluble or sample-suspendable reagents. These can be dissolved or suspended in the liquid sample as it flows in or through it and react with the analyte in the sample or with other sample components.

The different zones in the sample channel can also differ in that there are zones with capillary activity and zones without. In addition, zones with high hydrophilicity and low hydrophilicity can be included. The individual zones can pass into one another almost seamlessly or be separated from one another by certain barriers, for example valves, in particular non-closing valves such as geometric valves or hydrophobic barriers.

The reagents in the sample channel are preferably in dried or lyophilized form. However, it is also possible for reagents to be present in liquid form in the test element according to the invention.

The reagents can be introduced into the test element in a manner known per se. The test element preferably contains at least two layers, a base layer into which the fluidic structures are introduced and a cover layer which generally contains no further structures apart from the inlet openings for liquids and the ventilation openings. The introduction of reagents during the manufacture of the test device usually takes place before the upper part of the test element (top layer) is applied to the lower part (bottom layer). At this point in time, the fluidic structures in the lower part are open, so that the reagents can easily be dosed in liquid or dried form. The reagents can be introduced, for example, by printing or dispensing. It is also possible to introduce the reagents into the test element by placing them in the test element, impregnated in absorbent materials such as paper, fleece or membranes. After placing the reagents and inserting the absorbent materials, for example the porous, absorbent matrix (membrane) and optionally other absorbent materials (waste fleece, etc.), the upper and lower parts of the test element are connected to one another, for example clipped, welded, glued and the like.

Alternatively, it is possible that the bottom layer also has the inlet openings for liquids and the ventilation openings in addition to the fluidic structures. In this case, the cover layer can be formed entirely without openings, possibly with the exception of a central recess for accommodating a drive unit. Especially in this case, the upper part can simply consist of a plastic film that is glued to the lower part or welded to it.

The sample channel usually contains a zone for separating particulate components from the liquid sample. In particular, if blood or other body fluids with cellular components are used as the sample material, this zone serves to separate the cellular sample components. By separating the red blood cells (erythrocytes), in particular, almost colorless plasma or serum can be obtained from blood, which is generally more suitable for subsequent visual or optical detection methods than the strongly colored blood.

The cellular sample components are preferably separated off by centrifugation, i. H. by rapidly rotating the test element after filling it with a liquid sample. For this purpose, the test element according to the invention contains suitably dimensioned and geometrically designed channels and/or chambers. In particular, the test element for the separation of cellular blood components contains an erythrocyte collection zone (erythrocyte chamber or erythrocyte trap) and a serum or plasma collection zone (serum or plasma chamber).

In order to control the flow of the sample liquid in the test element, this can have valves, especially so-called non-closing (non-closing) or geometric valves or hydrophobic barriers. These valves serve as capillary stops. They can ensure that a targeted temporal and spatial control of the sample flow through the sample channel and the individual zone of the test element is possible.

In particular, the sample channel can have a sample dosing zone that allows the sample—which was initially applied in excess—to be measured precisely. In a preferred embodiment, the sample dosing zone extends from the sample application opening via a corresponding piece of a sample channel to a valve in the fluidic structure, in particular a geometric valve or a hydrophobic barrier. The sample application opening can initially take up an excess of sample material. The sample flows either driven by capillary forces or driven by centrifugation from the sample application zone into the channel structure and fills it up to the valve. Excess sample initially remains in the sample application zone. Only when the channel structure is filled up to the valve is a sample excess chamber adjacent to the sample application zone and branching off from the sample channel filled, for example by capillary forces or by centrifuging the test element. It must be ensured that the sample volume to be measured is not initially transported across the valve by selecting a suitable valve. As soon as excess sample is caught in the corresponding overflow chamber, there is a precisely defined sample volume between the valve of the sample channel on the one hand and the entrance to the sample overflow chamber on the other side. By applying external forces, in particular by starting another centrifugation, this defined sample volume is now moved beyond the valve. All fluidic areas that are downstream of the valve and come into contact with the sample are now initially filled with a precisely defined sample volume.

The sample channel also has an inflow for other liquids apart from the sample liquid. For example, a second channel can open into the sample channel, the z. B. can be filled with a washing or reagent liquid.

The system according to the invention made up of a measuring device and a test element is used to determine an analyte in a liquid sample. The measuring device contains, among other things, at least one drive for rotating the test element and evaluation optics for evaluating the visual or optical signal of the test element.

The optics of the measuring device can preferably be used for fluorescence measurement with spatially resolved detection. With two-dimensional, i. H. flat evaluation optics, an LED or a laser is typically used to illuminate the detection area of the test element and, if necessary, to excite optically detectable markings. The optical signal is detected using CMOS or CCD (typically with 640 × 480 pixels). The beam path is direct or folded (e.g. via mirrors or prisms).

In the case of anamorphic optics, the illumination or excitation is typically effected by means of an illumination line, which illuminates the detection area of the test element, preferably perpendicular to the detection and control lines. The detection can take place here via a diode line. In this case, the rotational movement of the test element can be used for the illumination and evaluation of the 2nd dimension in order to scan with the diode line over the flat area of the test element to be evaluated.

A DC motor with encoder or a stepper motor can be used as a drive for rotating and positioning the test element.

The temperature of the test element in the device is preferably controlled indirectly, for example by heating or cooling the plate on which the disc-shaped test element rests in the device. The temperature is preferably measured without contact.

The method according to the invention serves to detect an analyte in a liquid sample. The sample is first placed in the sample application opening of the test element. The test element is then rotated—preferably about its preferably central axis; however, it is also possible to carry out the method according to the invention in such a way that the rotation takes place around another axis, which may also be located outside of the test element. The sample is transported from the sample application opening to the end of the capillary-active zone, which is a porous, absorbent matrix, away from the axis. The rotation of the test element is then slowed down or stopped to such an extent that the sample or a material obtained from the sample as it flows through the test element (e.g. a mixture of the sample with reagents, a sample modified by pre-reactions with reagents from the test element, one of certain components liberated sample such as serum or plasma from whole blood after separating the erythrocytes, etc.) is transported from the end of the capillary-active zone that is remote from the axis to the end near the axis, which is a porous, absorbent matrix. Finally, the analyte is detected visually or optically in the capillary-active zone, which is a porous, absorbent matrix, or in a zone downstream of it.

The start of the migration of the sample (or a material obtained from the sample) through the capillary-active zone can be precisely timed and controlled by deliberately slowing down or stopping the rotation of the test element. Only when the amount of capillary force (suction force) in the capillary-active zone exceeds the amount of the counter-acting centrifugal force is it possible for the sample to move into and through the capillary-active zone. The liquid transport in the capillary-active zone can be started in a targeted manner. For example, a possible pre-reaction or pre-incubation of the sample or a tempering of the sample can be awaited before the rotation of the test element is slowed down or stopped to such an extent that the sample can flow into the capillary-active zone.

The transport of the sample (or a material obtained from the sample) through the capillary-active zone can be specifically slowed down or stopped by rotating the test element again about its preferably central axis. The centrifugal forces occurring during rotation counteract the capillary force, which moves the sample liquid from the end of the capillary-active zone that is remote from the axis to the end that is close to the axis. So is a targeted Control, in particular slowing down, of the flow rate of the sample in the capillary-active zone is possible, up to and including reversing the direction of flow. In this way, for example, the dwell time of the sample in the capillary-active zone can be controlled.

In particular, it is therefore possible, with the test element and method according to the invention, to reverse the direction of migration of the liquid sample and/or the other liquid through the capillary-active zone by rotating the test element, whereby this is also possible several times in order to avoid moving the to reach liquid. Through a targeted interplay of capillary forces, which transport the liquid in the capillary-active zone from the outside (i.e. from the end remote from the axis) inwards (i.e. to the end close to the axis), and oppositely directed centrifugal forces, it is possible, among other things, to increase the binding efficiency of the binding reactions in the capillary-active zone to increase, to better dissolve soluble reagents and to mix them with the sample or other liquids, or to increase the washing efficiency ("bound-free separation") in affinity assays.

In particular in connection with immunoassays, the detection can be carried out according to the sandwich assay principle or in the form of a competitive test.

It is also possible that, after the test element has been rotated, another liquid is applied to the test element, which is transported after the sample from the end of the capillary-active zone that is remote from the axis to the end near the axis.

The further liquid can in particular be a buffer, preferably a washing buffer, or a reagent liquid. By adding the further liquid, an improved signal-to-background ratio can be achieved in comparison to conventional test strips, particularly in connection with immunoassays, since the addition of the liquid can be used as a kind of washing step after the bound-free separation.

The invention has the following advantages:
The combination of liquid transport by means of centrifugal forces and by means of suction forces in capillary-active zones, in particular in porous, absorbent matrix materials, allows liquid flows to be precisely controlled. According to the invention, the capillary-active zone, which is a porous, absorbent matrix, transports the liquid from an end remote from the axis to an end close to the axis, ie from the periphery of the disk-shaped test element in the direction of the axis of rotation. The centrifugal force, which can also be used to move the liquids, counteracts this direction of transport. By specifically controlling the rotation of the test element (e.g. faster/slower rotation, switching the rotary movement on and off), it is therefore possible to slow down or to stop, so that targeted and defined reaction conditions can be maintained. At the same time, the use of the porous, absorbent matrix, which essentially serves as a capture matrix for bound-free separation in immunoassays, allows sample components to be captured efficiently during the course of the immunoassay. In particular, the interplay of centrifugal and capillary forces (suction forces) makes it possible to move the sample back and forth over a reagent zone, in particular a zone with immobilized reagents (above all, a capture zone for heterogeneous immunoassays), without increased technical effort, and so on to ensure more effective dissolving of the reagents, mixing of the sample with reagents or scavenging of sample components on immobilized binding partners. At the same time, it is possible to eliminate depletion effects when binding sample components (above all analyte) to immobilized binding partners and thus increase the binding efficiency (i.e. sample parts depleted in analyte can be separated by analyte-rich sample parts are replaced). In addition, by moving liquids back and forth in the capillary-active zone, the most efficient possible use of the small liquid volumes can be achieved not only for Reaction purposes (here the sample volume is used in particular) but also for washing purposes, for example for better discrimination of bound and free label in the capture zone. This effectively minimizes the amount of sample, liquid reagents and wash buffer.

The preferably central position of the axis of rotation within the test element makes it possible to design both the test element itself and the associated measuring device as compactly as possible. With chip-shaped test elements, such as those used in figure 1 and 2 from US2004/0265171 are shown, the axis of rotation lies outside the test element. An associated turntable or rotor is therefore inevitably larger than in a test element with identical dimensions, but in which the axis of rotation is preferably arranged centrally within the test element, as is the case with the test elements according to the invention.

The invention is explained in more detail by the following examples and figures. Here, reference is made in each case to immunological sandwich assays. However, the invention is not limited to this. It can also be applied to other types of immunoassays, in particular also to competitive immunoassays, or other types of specific binding assays (for example those that use sugar and lectins, hormones and their receptors, or complementary nucleic acid pairs as binding partners). Typical representatives of these specific binding assays are known per se to those skilled in the art (in connection with immunoassays, reference is expressly made here to figures 1 and 2 and the associated descriptive passages of the document U.S. 4,861,711 referenced) and can be readily applied to the present invention. figure 1 shows a schematic representation of a plan view of an embodiment of a test element that is not according to the invention. For the sake of clarity, only the layer of the test element that contains the fluidic structures is shown. The embodiment shown contains only one opening for the introduction of sample and / or washing liquid. In this embodiment, the interfering sample components are separated after the sample has been brought into contact with reagents.

figure 2 shows schematically a further embodiment of a test element not according to the invention. Here, too, only the structure that has the fluidic elements of the test element was depicted. In this embodiment of the test element there are two separate sample and wash buffer application ports. The cellular sample components are already separated here before the sample is brought into contact with reagents.

figure 3 shows a variant of the embodiment according to the invention figure 1 in a schematic representation. Here, too, the cellular sample components are separated after the sample has been brought into contact with reagents. According to the invention, the structure according to figure 3 a separate supply for washing liquid.

figure 4 shows a further preferred embodiment of a test element according to the invention in a schematic view analogously figure 2 .

figure 5 represents a minor further development of the test element according to the invention figure 3 represents. In contrast to the embodiment according to figure 3 contains figure 5 a different geometric arrangement of the waste fleece and a different type of valve at the end of the sample dosing section.

figure 6 shows schematically a top view of a further development of the test element according to the invention according to FIG figure 5 . In contrast to the embodiment according to figure 5 contains the embodiment according to figure 6 a fluidic structure for taking up excess sample.

figure 7 is a schematic representation of a further variant of the test element according to the invention figure 3 . Functionally, the fluidic structures are essentially analogous to those of figure 3 . However, they are geometrically aligned and designed differently.

figure 8 shows schematically a further preferred embodiment of the test element according to the invention. The structures in figure 8 essentially correspond to the functions provided by the test element figure 4 are already known.

figure 9 shows schematically a top view of an alternative to the test element according to FIG figure 6 . In contrast to the embodiment according to the invention figure 6 contains the embodiment according to figure 9 a sample application opening remote from the axis, which initially brings the sample closer to the center of the test element via a capillary, ie into an area close to the axis.

figure 10 shows the typical curve shape for troponin T measurements in whole blood samples (concentration of troponin T in ng/ml plotted against the signal strength (counts)). The samples were spiked with recombinant troponin T to the respective concentration.

The data pertains to Example 2 and was determined using test elements according to figure 6 / Sample 1 received.

1

scheibenförmiges Testelement (Disk)

2

Substrat (z. B. ein- oder mehrteilig, Spritzguss, gefräst, aus Schichten aufgebaut etc.)

3

zentrale Aussparung (Antriebsloch)

4

Probenaufgabeöffnung

5

Probendosierzone (Dosierabschnitt des Kanals)

6

Kapillarstopp (z. B. hydrophobe Barriere, geometrisches/nicht-schließendes Ventil)

7

Behälter für Probenüberschuss

8

Kapillarstopp (z. B. hydrophobe Barriere, geometrisches/nicht-schließendes Ventil)

9

Kanal

10

Serum-/Plasmasammelzone (Serum-/Plasmakammer)

11

Erythrozytensammelzone (Erythrozytenkammer)

12

poröse, saugfähige Matrix (Membran)

13

Waste (Vlies)

14

Kapillarstopp (z. B. hydrophobe Barriere, geometrisches/nicht-schließendes Ventil)

15

Kanal

16

Zugabeöffnung für weitere Flüssigkeiten, z. B. Waschpuffer

17

Entlüftungsöffnung

18

Dekantierkanal

19

Kapillarstopp (z. B. hydrophobe Barriere, geometrisches/nicht-schließendes Ventil)

20

Auffangreservoir

21

Kapillarkanal

The numbers and abbreviations in the figures have the following meaning:

1

disc-shaped test element (disk)

2

Substrate (e.g. one or more parts, injection molding, milled, made up of layers, etc.)

3

central recess (drive hole)

4

sample application port

5

sample dosing zone (dosing section of the channel)

6

Capillary stop (e.g. hydrophobic barrier, geometric/non-closing valve)

7

Container for excess sample

8th

Capillary stop (e.g. hydrophobic barrier, geometric/non-closing valve)

9

channel

10

Serum/plasma collection zone (serum/plasma chamber)

11

Erythrocyte collection zone (erythrocyte chamber)

12

porous, absorbent matrix (membrane)

13

waste

14

Capillary stop (e.g. hydrophobic barrier, geometric/non-closing valve)

15

channel

16

Addition opening for further liquids, e.g. B. Wash Buffer

17

vent hole

18

decantation channel

19

Capillary stop (e.g. hydrophobic barrier, geometric/non-closing valve)

20

collection reservoir

21

capillary channel

the Figures 3 to 8 show different preferred embodiments of a test element (1) according to the invention. the figures 1 , 2 and 9 show alternative embodiments of a test element that are not according to the invention. The substrate (2) containing the fluidic structures and the central recess (drive hole 3) is essentially shown in each case. In addition to the substrate, which can be one or more parts, for example, and can be constructed by injection molding, milling or by laminating corresponding layers, the disk-shaped test element (1) according to the invention also usually contains a cover layer, which is shown in the figures for clarity is not shown for the sake of it. In principle, the cover layer can also have structures, but as a rule it will not have any structures apart from the openings for the samples and/or other liquids to be applied to the test element. The cover layer can also be designed without any openings, for example in the form of a film which is connected to the substrate and closes off the structures located therein.

The embodiments in the Figures 1 to 9 are shown show fluidic structures that largely fulfill the same functions, even if they differ in detail from embodiment to embodiment. The basic structure and the basic function should therefore be described here in more detail based on the embodiment figure 1 be explained. The embodiments according to Figure 2 to 9 are then explained in more detail based on the specific differences between them in order to avoid unnecessary repetition.

figure 1 shows a first embodiment of a disc-shaped test element (1) not according to the invention. The test element (1) contains a substrate (2) which contains the fluidic, microfluidic and chromatographic structures. The substrate (2) is covered (not shown) by a corresponding counterpart (top layer) which contains sample application and ventilation openings which correspond to the structures in the substrate (2). Both the cover layer and the substrate (2) have a central recess (3) which, in cooperation with a corresponding drive unit in a measuring device, enables the disk-shaped test element (1) to rotate. Alternatively, it is possible that the test element (according to one of Figures 1 to 9 ) has no such central recess (3) and the drive is rotated via a drive unit of the measuring device designed according to the outer contours of the test element, for example a rotating plate, in which the test element is placed in a depression corresponding to its shape.

Sample liquid, in particular whole blood, is fed to the test element (1) via the sample application opening (4). Driven by capillary forces and/or centrifugal forces, the sample liquid fills the sample dosing zone (5). The sample dosing zone (5) can also contain the dried reagents. It is delimited by the capillary stops (6 and 8), which can be designed, for example, as a hydrophobic barrier or as a geometric/non-closing valve. The delimitation of the sample metering zone (5) by the capillary stops (6, 8) ensures that a defined sample volume is taken up and passed on to the fluidic zones that are downstream of the sample metering zone (5). When the test element (1) rotates, any excess sample is removed from the sample application opening (4) and the sample dosing zone (5) is transferred to the sample excess container (7), while the measured amount of sample is transferred from the sample dosing zone (5) to the channel (9).

With appropriate rotation speeds, the separation of red blood cells and other cellular sample components is started in channel (9). The reagents contained in the sample dosing zone (5) are already dissolved in the sample when it enters the channel (9). The entry of the sample into the channel (9) via the capillary stop (8) leads to the reagents in the sample being mixed.

The possibility of temporal control of the rotation processes, which is possible with the test element according to the invention, allows a targeted control of the dwell times and thus the incubation time of the sample with reagents and the reaction times.

During the rotation, the reagent-sample mixture is directed into the fluidic structures (10) (serum/plasma collection zone) and (11) (erythrocyte collection zone). Due to the centrifugal forces acting on the reagent-sample mixture, plasma or serum is separated from red blood cells. The red blood cells collect in the erythrocyte collection zone (11), while the plasma essentially remains in the collection zone (10).

In contrast to test elements that use membranes or nonwovens for separating particulate sample components (e.g. glass fiber nonwovens or asymmetrically porous plastic membranes for separating red blood cells from whole blood, generally referred to as blood-separating membranes or nonwovens), the sample volume can be used much more effectively with the test elements according to the invention, since there are practically no dead volumes (e.g. volume of the fiber interstices or pores) from which the sample cannot be taken again. In addition, these prior art blood-separating membranes and webs tend to be undesirable in some cases Adsorption of sample components (e.g. proteins) or destruction (lysis) of cells, which is also not observed with the test elements according to the invention.

If the rotation of the test element (1) is stopped or slowed down, the reagent-plasma mixture (in which, in the case of an immunoassay, sandwich complexes of analyte and antibody conjugates have formed when the analyte is present) is absorbed by the suction effect of the porous-absorbent matrix (12) added to and passed through it. In the case of immunoassays, the analyte-containing complexes are captured in the detection zone by the immobilized binding partners contained in the membrane (12) and unbound, labeled conjugate is bound in the control zone. The fleece (13) adjoining the porous, absorbent matrix supports the movement of the sample through the membrane (12). The fleece (13) also serves to hold the sample after it has flowed through the membrane (12).

After the liquid sample has flowed through the fluidic structure of the test element (1) from the sample application opening (4) to the fleece (13), the washing buffer is pipetted into the sample application opening (4) in a subsequent step. Using the same combination of capillary, centrifugal and chromatographic forces, the wash buffer flows through the corresponding fluidic structures of the test element (1) and washes in particular the membrane (12), where the bound analyte complexes are now located and thus removes excess reagent residues. The washing step can be repeated one or more times in order to improve the signal-to-noise ratio. This allows the detection limit for the analyte to be optimized and the dynamic measuring range to be increased.

The sample channel, in which the liquid sample in the test element (1) is transported from the sample application opening (4) to the first end of the membrane (12) remote from the axis, in the present case comprises the sample dosing zone (5), the capillary stop (8), the channel ( 9), the serum/plasma collection zone (10) and the erythrocyte chamber (11). In other In some embodiments, the sample channel can consist of more or fewer individual zones/areas/chambers.

the Figures 3 , 5 , 6 , 7 and 9 show essentially analogous embodiments figure 1 . figure 3 differs from figure 1 in that, on the one hand, there is no container for excess sample (7) adjoining the sample application opening (4) and there is no capillary stop at the end of the sample metering section (5) (ie metered sample application is required here) and, on the other hand, in that, according to the invention, a separate metering opening ( 16) for other liquids, e.g. B. washing buffer, and an associated channel (15) are present, which can transport the buffer to the membrane (12). The transport of the buffer to the membrane (12) can be based on capillary forces or centrifugal forces.

The embodiment according to figure 5 is largely identical to the embodiment according to FIG figure 3 . The two embodiments differ only in the shape of the waste fleece (13) and in that the test element according to FIG figure 5 a capillary stop (8) at the end of the sample dosing section (5).

The embodiment according to figure 6 is again essentially identical to the embodiment according to FIG figure 5 and differs from this by the additional presence of a container for excess sample (7) in the area between the sample dosing opening (4) and the sample dosing zone (5). No dosed application of the sample is required here (analogous to figure 1 ).

The embodiment of the test element (1) according to the invention figure 7 essentially corresponds to the test element (1) of figure 6 . Both embodiments have the same fluidic structures and functions. Only the arrangement and geometric design is different. The embodiment according to figure 7 has additional ventilation openings (17), which due to the different dimensions of the fluidic structures compared to figure 6 are necessary to fill the To allow structures with samples or washing liquid. Channel (9) is designed here as a thin capillary which is only filled when the test element rotates (ie overcoming the capillary stop (8) is only possible by means of centrifugal force). It is already in accordance with the test element (1) during the rotation figure 7 possible to discharge collected plasma from the erythrocyte collection zone 11; the decanting unit 18, which finally opens into the serum/plasma collection zone 10, is used for this purpose.

The embodiment of a test element (1) according to the invention figure 9 essentially corresponds to the test element (1) of figure 6 . Both embodiments have the same fluidic structures and functions. Only the arrangement and geometric design is different. The non-inventive embodiment according to figure 9 basically has a sample application opening (4) further to the outside, i.e. away from the axis. This can be advantageous if the test element (1) has already been placed in a measuring device for filling with sample. In this case, the sample application opening (4) can be made accessible to the user more easily than is the case with test elements according to FIG Figure 1 to 8 is possible where the sample application opening (4) is arranged close to the axis (ie away from the outer edge of the test element).

In contrast to the embodiment according to figure 1 , 3 , 5 , 6 , 7 and 9 takes place in the embodiments according to figure 2 , 4 and 8th the separation of the cellular sample components from the sample liquid takes place before the sample comes into contact with reagents. This has the advantage that the use of whole blood or plasma or serum as sample material does not lead to different measurement results, since plasma or serum always comes into contact with the reagents first and the dissolution/incubation/reaction behavior should therefore be practically the same . Also in the embodiments according to figure 2 , 4 and 8th the liquid sample is first applied to the test element (1) via the sample application opening (4). The sample is then transported further from the sample application opening (4) into the channel structures by capillary forces and/or centrifugal forces. In the embodiments according to figure 2 and 4 will the sample after transferred to the sample application opening (4) in a sample dosing section (5) and then causes a serum/plasma separation from the whole blood by rotation. The undesired cellular sample components, essentially erythrocytes, collect in the erythrocyte trap (11), while serum or plasma collect in zone (10). The serum is removed from the zone (10) via a capillary and transported further into the channel structure (9), where dried reagents are housed and are dissolved when the sample flows in. By rotating the test element (1) again, the sample-reagent mixture from the channel structure (9) can overcome the capillary stop (14) and thus reach the membrane (12) via the channel (15). When the rotation slows down or stops, the sample-reagent mixture is transported across the membrane (12) into the waste fleece (13).

The embodiments according to figure 2 and figure 4 differ in that in figure 2 a container for excess sample (7) is provided, while the embodiment according to figure 4 does not provide such a function. As in the embodiment according to figure 3 a dosed application of the sample is advisable here.

figure 8 shows a variant of the embodiments according to FIG figures 2 and 4 . Here, the sample is transferred by centrifugation into an erythrocyte separation structure (10, 11) immediately after the sample application opening (4) after passing through a first geometric valve (19). The area marked (10) serves as a serum/plasma collection zone (10) from which serum or plasma freed from cells is passed on via a capillary channel (21) after centrifugation. Chamber (20) serves as a collection reservoir for excess serum or plasma, which may flow out of the serum/plasma collection zone (10) after the sample metering section (5) has been completely filled. All other functions and structures are analogous to the Figures 1 to 7 .

By specifically designing the hydrophilic or hydrophobic properties of the surfaces of the test element (1), it can be achieved that the sample liquid and/or Washing liquids either only with the help of rotation and the resulting centrifugal forces or by a combination of centrifugal forces and capillary forces. The latter requires at least partially hydrophilized surfaces in the fluidic structures of the test element (1).

As above in connection with figure 1 already described, the test elements according to the invention according to figures 6 , 7 and, 8th an automatic functionality that allows a relatively accurate measurement of a sample aliquot from a sample applied in excess to the test element (so-called "metering system"). It essentially comprises the elements 4, 5, 6 and 7 of the test elements (1) shown. Sample liquid, in particular whole blood, is fed to the test element (1) via the sample application opening (4). Driven by capillary forces and/or centrifugal forces, the sample liquid fills the sample dosing zone (5). The sample dosing zone (5) can also contain the dried reagents. It is delimited by the capillary stops (6 and 8), which can be designed, for example, as a hydrophobic barrier or as geometric/non-closing valves. The delimitation of the sample metering zone (5) by the capillary stops (6, 8) ensures that a defined sample volume is taken up and passed on to the fluidic zones that are downstream of the sample metering zone (5). When the test element (1) rotates, any excess sample is transferred from the sample application opening (4) and the sample dosing zone (5) into the container for excess sample (7), while the measured amount of sample from the sample dosing zone (5) flows into the channel (9 ) is transferred. Alternatively, it is also possible to use other forces instead of the force generated by rotation that moves the sample, e.g. B. by applying an overpressure on the sample inlet side or a negative pressure on the sample outlet side. Consequently, the metering system shown is not necessarily tied to rotatable test elements, but can also be used in other test elements.

Similar metering systems are out, for example U.S. 5,061,381 known. This document also describes a system in which excess sample liquid can be applied to a test element. The measuring of a relatively accurate sample aliquot, which is then further processed in the test element, is also carried out here by the interaction of a dosing zone ("metering chamber") and a zone for the excess sample ("overflow chamber"), whereby these two zones - unlike in the present invention - by a contact that is narrow, but always allows liquid exchange at least during filling. When the test element is filled, the sample liquid is immediately separated into a part that is conducted through a wide channel into the "metering chamber" and a part that flows through a narrow channel into the "overflow chamber". After the "metering chamber" has been completely filled, the test element is set in rotation and any excess sample is drained into the "overflow chamber", so that only the desired, measured sample volume remains in the "metering chamber", which is then processed further.

A disadvantage of the design of the metering system according to U.S. 5,061,381 is that with sample volumes that are applied to the test element and that correspond exactly to the minimum volume or are only slightly larger than the minimum volume, there is a risk that the dosing zone will be underdosed, since a proportion of the sample always flows unhindered into the "overflow chamber" flows.

This problem is solved in the configuration of the metering system proposed here in that a capillary stop (hydrophobic barrier or a geometric or non-closing valve) is arranged between the dosing zone and the zone for the excess sample. Therefore, when the test element is filled with sample, the sample is initially fed practically exclusively into the dosing zone. The capillary stop prevents the sample from flowing into the zone for sample excess before the sample dosing zone is completely filled. Even with sample volumes that are applied to the test element and which correspond exactly to the minimum volume or are only slightly larger than the minimum volume, this ensures that the sample dosing zone is completely filled.

example 1 Production of a test element according to FIG 1.1. Production of the substrate (2)

• Kapillare zwischen 4 und 5: t = 500 µm
• Nr.7: t= 700 µm
• Nr.5: t= 150 µm; V= 26,5 mm³
• Nr.8: t= 500 µm
• Nr.9: t= 110 µm
• Nr.10: t= 550 µm
• Nr.11: t= 130 µm; V= 15 mm³
• Nr.15: t= 150 µm; V= 11,4 mm³

A substrate (2) according to FIG figure 6 manufactured (dimensions approx. 60 × 80 mm ² ). The individual channels and zones (fluidic structures) have the following dimensions (depth of the structures (t) and possibly their volume (V); the numbers refer to figure 6 and the structures shown therein):

• Capillary between 4 and 5: t = 500 µm
• No.7: t= 700 µm
• No.5: t= 150 µm; V= 26.5mm ³
• No.8: t= 500 µm
• No.9: t= 110 µm
• No.10: t= 550 µm
• No.11: t= 130 µm; V= 15mm ³
• No. 15: t= 150 µm; V= 11.4mm ³

A transition from less deep to deeper structures is generally only possible for liquids in the fluidic structures if an external force (e.g. centrifugal force) acts. Such transitions act as geometric (non-closing) valves.

In addition to the fluidic structures (see above), the substrate (2) also has the sample and buffer feed openings (4, 16), ventilation openings (17) and the central recess (3).

The surface of the substrate (2), which has the fluidic structures, can then be cleaned and rendered hydrophilic by means of plasma treatment.

1.2. Bringing in the reagents

Some of the reagents required for analyte detection (e.g. biotinylated anti-analyte antibodies and anti-analyte antibodies marked with a fluorescent label) are introduced as a solution by means of piezo metering alternately as punctiform reagent spots into the sample metering section (5) and then dried. so that practically its entire inner surface is covered with reagents.

The reagent solutions are composed as follows: Biotinylated Antibody: 50mM Mes pH 5.6; 100 µg/ml biotinylated monoclonal anti-troponin T antibodies Labeled antibodies 50 mM Hepes pH 7.4, with squaric acid derivative fluorescent dye JG9 (embedded in polystyrene latex particles) fluorescently labeled anti-troponin T monoclonal antibody (0.35 percent solution)

1.3. Inserting the membrane (12)

In a corresponding recess in the substrate (2), the porous matrix (12) (nitrocellulose membrane on plastic carrier film; 21 × 5 mm ² ; reinforced with 100 micron PE film cellulose nitrate membrane (type CN 140 from Sartorius, Germany)), in which an analyte detection line (polystreptavidin) and a control line (polyhapten) were introduced by means of line impregnation (see below), inserted and, if necessary, fixed with double-sided adhesive tape.

An aqueous streptavidin solution (4.75 mg/ml) is applied by line dosing to the cellulose nitrate membrane described above. For this purpose, the dosing is selected (dosing quantity 0.12 ml/min, web speed 3 m/min) that a line with a width of approx. 0.4 mm arises. This line serves to identify the analyte to be determined and contains approx. 0.95 µg streptavidin per membrane.

At a distance of about 4 mm downstream from the streptavidin line, an aqueous 0.3 mg/ml troponin T polyhapten solution is applied under identical dosing conditions. This line serves as a function control of the test element and contains approx. 0.06 µg polyhapten per test.

1.4. Applying the cover

The cover (foil or injection-molded part without fluidic structures, which can optionally be made hydrophilic) is then applied and optionally permanently connected to the substrate (2), preferably glued, welded or clipped.

1.5. Insertion of the waste fleece (13)

Finally, the substrate is turned and the waste fleece (13) (13 × 7 × 1.5 mm ³ fleece made of 100 parts glass fiber (diameter 0.49 to 0.58 µm, length 1000 µm) and 5 Parts of polyvinyl alcohol fibers (Kuralon VPB 105-2 from Kuraray) with a basis weight of about 180 g/m ² ) are inserted, which is then fixed in the substrate (2) by means of an adhesive tape.

The quasi-self-dosing sample receiving unit (comprising the sample application opening (4), the sample dosing section (5) and the structures delimiting it (capillary stop (8) and container for excess sample (7)) ensures that, regardless of the amount applied to the test element (1). sample amount (if it exceeds a minimum volume (in this example 27 µl)) when using different test elements, the same sample amounts can be used reproducibly.

By distributing the reagents throughout the sample dosing section (5), preferably in the form of alternating reagent spots (i.e. small, almost punctiform reagent areas), in combination with rapid filling of the sample dosing section (5) with sample, a homogeneous dissolution of the reagents in the entire sample volume is achieved , especially if the filling is much faster than the release. In addition, the reagents are practically completely dissolved, so that here again an increased reproducibility compared to conventional test elements based on absorbent materials (test strips, bio-disks with reagent pads, etc.) is observed.

example 2 Detection of troponin T using the test element from example 1

27 μl of whole blood to which different amounts of recombinant troponin T have been added are applied to the test element according to Example 1. The test element is then processed further using the procedure given in Table 1 and finally the fluorescence signals for different concentrations are measured. Table 1: Measurement process Time (min:sec) Duration (min:sec) Rotation with revolutions per minute action 00:00 01:00 0 apply 27 µl sample; dissolving the reagents 01:00 02:00 5000 Erythrocyte Separation and Incubation 03:00 01:00 800 Chromatography (generate signal) 04:00 00:10 0 Add 12 µl wash buffer ¹⁾ . 04:10 02:00 800 Wash Buffer Transport and Chromatography 06:10 00:10 0 Add 12 µl wash buffer ¹⁾ . 06:20 02:00 800 Wash Buffer Transport and Chromatography 08:20 00:10 0 Add 12 µl wash buffer ¹⁾ . 08:30 02:00 800 Wash Buffer Transport and Chromatography 10:30 a.m 0 Measure ¹⁾ 100mM Hepes, pH 8.0; 150mM NaCl; 0.095% sodium azide.

The measurement data are in figure 10 played back. The respective measurement signals (in counts) are plotted against the concentration of recombinant troponin T (c(TnT)) in [ng/ml]). The actual troponin T concentration in the whole blood samples was determined using the "Roche Diagnostics Elecsys Troponin T Test" reference method.

Compared to conventional immunochromatographic troponin T test strips such. B. Cardiac troponin T from Roche Diagnostics, the detection limit for the quantitatively evaluable measuring range with the test element according to the invention is shifted down (cardiac troponin T: 0.1 ng / ml; invention: 0.02 ng / ml) and the dynamic measuring range extended upwards (cardiac troponin T: 2.0 ng/ml; invention: 20 ng/ml). At the same time, the test elements according to the invention show improved precision.

Claims (11)

1. A test element (1) for detecting an analyte in a liquid sample which is substantially disk-shaped and rotatable about an axis perpendicular to the plane of the disk-shaped test element, containing

   - a sample application opening (4) for applying a liquid sample,

   - a capillary-active zone (12) which is a porous, absorbent matrix (12), wherein the capillary-active zone (12) contains one or more zones with immobilized reagents, wherein the immobilized reagents are specific binding reagents which are suitable to selectively capture the analyte or species derived from and related to the analyte from the sample flowing through the capillary-active zone (12) and which are present in the form of lines, dots, patterns immobilized in or on the material of the capillary-active zone (12), having a first end remote from the axis and a second end near to the axis, and

   - a sample channel (9) extending from the sample application opening (4) via an area near to the axis to the first end of the capillary-active zone (12) that is remote from the axis;

   characterized in that

   the first end of the capillary-active zone (12) that is remote from the axis is in contact with the sample channel (9); and

   the sample application opening (4) is near to the axis and the sample channel (9) extends from the sample application opening (4) near to the axis to the first end of the capillary-active zone (12) that is remote from the axis; and

   the sample channel has an inlet for further liquids except for the sample liquid; and wherein said inlet has a separate addition opening (16) for further liquids, preferably washing buffer, and an associated channel (15) capable of transporting the buffer to the capillary-active zone (12), wherein

   the separate addition opening (16) is arranged closer to the axis about which the test element is rotatable than the area of the sample channel (9) which is directly connected to the first end of the capillary-active zone (12) that is remote from the axis; and

   the associated channel (15) directly connects the separate addition opening (16) to the area of the sample channel (9) which is directly connected to the first end of the capillary-active zone (12) that is remote from the axis.
2. The test element (1) according to claim 1, characterized in that the porous, absorbent matrix (12) is a paper, a membrane, or a fleece.
3. The test element (1) according to one of the preceding claims, characterized in that the capillary-active zone (12) with the second end near to the axis is in contact with a further absorbent material (13) or an absorbent structure capable of taking up liquid from the capillary-active zone (12).
4. The test element (1) according to one of the preceding claims, characterized in that the sample channel (9) contains zones of different dimensions and/or for different functions, in particular a zone with soluble reagents and/or a sample dosing zone (5) and/or a zone for separating particulate components from the liquid sample.
5. The test element (1) according to one of the preceding claims, characterized in that the sample channel (9) contains geometric valves or hydrophobic barriers (6, 8, 14, 19).
6. The test element (1) according to one of claims 1 to 5, characterized in that the sample application opening (4) is in contact with a sample dosing zone (5) and a zone (7) for sample excess, wherein a capillary stop (6) is present between the sample dosing zone (5) and the zone (7) for sample excess.
7. A method for detecting an analyte in a liquid sample, wherein

   - the sample is applied into the sample application opening (4) of the test element (1) according to one of claims 1 to 6,

   - the test element (1) is rotated such that the sample is transported to the end of the capillary-active zone (12) that is remote from the axis;

   - the rotation of the test element (1) is decelerated or stopped, respectively such that the sample or a material recovered from the sample when flowing through the test element (1) is sucked from the end remote from the axis to the end near to the axis of the capillary-active zone (12); and

   - the analyte is visually or optically detected in the capillary-active zone (12) or a zone downstream thereof.
8. The method according to claim 7, characterized in that
   after rotation of the test element (1) a further liquid is applied to the test element (1) which is sucked after the sample from the end remote from the axis to the end near to the axis of the capillary-active zone (12).
9. The method according to claim 7 or 8, characterized in that migration of the liquid sample and/or the further liquid through the capillary-active zone (12) is selectively decelerated or stopped by the rotation of the test element (1).
10. The method according to one of claims 7 to 9, characterized in that the migration direction of the liquid sample and/or the further liquid through the capillary-active zone (12) is reversed by the rotation of the test element (1).
11. A system for determining an analyte in a liquid sample containing a test element (1) according to one of claims 1 to 6 and a measuring device, wherein the measuring device has

    - at least one drive for the rotation of the test element (1); and

    - an evaluation optics for evaluating the visual or optical signal of the test element (1).

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