EP1916524A1 - Rotatable test element - Google Patents

Abstract

A disk-shaped, flat test element (1), rotatable about a (preferably central) axis perpendicular to the plane of the element, has a liquid sample introduction opening (4); a capillary-active region (12) (preferably a porous absorbent matrix) having a first end near and a second end spaced from the axis; and a sample channel (9) extending from near the axis to the first end of the capillary-active region. Independent claims are included for: (1) a variant of the test element, having a sample receiving opening with a sample dosing zone (5) contacting an excess sample zone (7), where a capillary stopper (6) is provided between the sample dosing and excess sample zones; (2) a method for determining an analyte in a liquid sample using the element, involving: applying the sample to the receiving opening; rotating the the element, so that the sample is transported to the first end of the capillary-active zone; slowing or stopping the rotation, so that the sample (or a material recovered from the sample on passage through the element) is sucked from the first end to the second end of the capillary-active zone; and visually or optically determining the analyte in the capillary-active zone or an adjacent zone; and (3) a system for determining an analyte in a liquid sample, comprising a test element as above and a measuring device including a drive for rotating the element and an optical system for evaluating the visual or optical signal from the element.

Description

The invention relates to a test element, which is substantially disc-shaped and flat and about a central axis which is perpendicular to the disc-shaped test element plane, rotatable, comprising a sample application port for discharging a liquid sample, a porous, absorbent matrix and a sample channel, of the Sample introduction opening to the porous, absorbent matrix is sufficient. Furthermore, the invention relates to a method for determining an analyte with the aid of the test element.

In principle, one can subdivide the systems, which are used for the analysis of liquid sample materials or sample materials, which can be converted into liquid form, into two classes: on the one hand there are analysis systems, which work exclusively with so-called wet reagents, on the other hand there are systems, which work with so-called dry reagents. Particularly in medical diagnostics, but also in environmental and process analysis, the former systems have prevailed in the field of permanently installed laboratories, while the latter systems are used primarily in the field of "on-site" analytics.

In the field of medical diagnostics, analysis systems with dry reagents, in particular in the form of so-called test carriers, eg. B. Test strips offered. Prominent examples of these are test strips for the determination of the blood glucose value or test strips for urinalysis. Such test carriers usually integrate several functions (eg the storage of reagents in dried form or, albeit less frequently, in solution, the separation of undesired sample constituents, in particular of red blood cells from whole blood, in immunoassays, the so-called Free separation, the dosing of sample volumes, the transport of sample liquid from outside a device into a device, the control of the sequence of individual reaction steps, etc.). The function of the sample transport is often accomplished by means of absorbent materials (eg papers or fleeces), by means of capillary channels or by application of external driving forces (such as pressure, suction) or by means of centrifugal force. Disc-shaped test carriers, so-called LabDiscs or optical BioDiscs, continue the idea of controlled sample transport by means of centrifugal force (centrifugal force). Such Disc-shaped, CompactDisc-like test carriers allow miniaturization through the use of microfluidic structures and at the same time the parallelization of processes by repeated application of identical structures for the parallel processing of similar analyzes from a sample or identical analyzes from different samples. Especially in the field of optical BioDiscs the integration of optically stored digital data for the identification of the test carriers 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 methods and measured using established evaluation technology. In the chemical and biochemical components of such optical BioDiscs usually a recourse to known chemical and biochemical components is possible. A disadvantage of the optical LabDiscs or Biodiscs based purely on centrifugal and capillary forces is that the immobilization of reagents is difficult and the accuracy of detection suffers. Especially in detection systems based on specific binding reactions, such. As immunoassays, lacks compared to conventional test strip systems, the volume component, especially in the so-called. Bound-Free separation.

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

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

Out WO 2005/009581 (Randall et al. ), optical bio-disks are known which contain absorbent membranes or papers for moving a sample liquid, separating particulate sample components, carrying reagents, and 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. Thereafter, the sample is again moved radially outward, ie away from the center of the bio-disk, and flows through a so-called Analysis membrane. The outward movement takes place via chromatography, which is supported by rotation of the bio-disk and the centrifugal force acting thereon on the sample.

US 2002/0076354 A1 (Cohen ) discloses bio-optical discs having, in addition to a channel system for the transport of a liquid sample, a so-called "capture layer". The latter can for example consist of nitrocellulose. The "interception layer" is traversed by means of centrifugal forces during rotation of the disk.

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

A disadvantage of the concepts of the prior art is that just for specific binding assays, such. As immunoassays 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 invention relates to a test element according to claim 1 or 14, a measuring system according to claim 18, the use thereof according to claim 19, as well as a method according to claim 15. Advantageous embodiments and preferred embodiments of the invention are the subject of the dependent claims.

The test element according to the invention is essentially disc-shaped and planar. It is rotatable about a central axis which is perpendicular to the disk-shaped test element plane. Typically, the test element is a circular disc, comparable to a compact disc. However, the invention is not limited to this form of the disc, but can readily be found in non-symmetrical or non-circular discs use.

As constituents, the test element first contains a sample introduction opening into which a liquid sample can be pipetted or introduced in another way. The Sample loading aperture can either be close to the axis (ie near the center of the disc) or far away from the axis (ie near the edge of the disc). In the event that the sample application opening is remote from the axis, the test element contains at least one channel, which can transfer the liquid sample from the off-axis position into an axis-near position by means of capillary forces.

The sample application opening can open directly into a sample channel. However, it is also possible for the sample introduction opening to first open into a reservoir located behind it, into which the sample flows before it continues to flow into the sample channel. By suitable dimensioning, it can be ensured that the sample flows from the sample application opening into the subsequent fluidic structures without any further action. For this purpose, a hydrophilization of the surfaces of the fluidic structures may be necessary and / or the use of structures that promote the formation of capillary forces. However, it is also possible to allow the filling of the fluidic structures of the test element according to the invention from the sample application opening only after the action of an external force, preferably a centrifugal force.

Furthermore, the test element contains a porous, absorbent matrix which accommodates at least part of the liquid sample. The matrix has an off-axis first end and an off-axis second end.

The test element also has a sample channel that extends from the sample introduction port to the off-axis first end of the porous absorbent matrix. At least once, the sample channel passes through an area close to the axis, which is closer to the central axis than the first end of the porous matrix, which is remote from the axis.

Essential to the test element of the present invention is that the porous, absorbent matrix has an axis-proximate second end. The off-axis first end of the porous, absorbent matrix 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 overpressure or underpressure. As soon as the liquid sample - possibly after taking up reagents and / or dilution media and / or precluding reactions - reaches the off-axis first end of the porous, absorbent matrix, it is taken up in the matrix and transported through the suction forces.

Typically, the porous, absorbent matrix is a paper, membrane or web.

The porous absorbent matrix typically contains one or more zones of immobilized reagents.

Typically, specific binding agents, such as specific binding partners, such as antigens, antibodies, (poly) haptens, streptavidin, polystreptavidin, and the like, are immobilized in the absorbent matrix. They serve to selectively capture from the sample flowing through the absorbent matrix the analyte or analyte-derived and related species. For example, in the case of immunoassays, an antibody to the analyte may be immobilized in the porous, bibulous matrix which will then capture the analyte (in this case, an antigen or hapten) from the sample and also immobilize it in the bibulous matrix. The analyte can be made detectable by further reactions, for example by further contacting with a labeled bindable partner, for example by a label which can be visually, optically or fluorescently detected.

In a preferred embodiment of the test element according to the invention, the porous, absorbent matrix with the second end close to the axis adjoins another absorbent material, so that this liquid can absorb from the matrix. Typically, the matrix and other material overlap slightly for this purpose. The other material serves on the one hand to support the suction of the matrix, on the other hand as a receiving zone for liquid, which has already passed through the matrix. The further material may consist of the same or different materials, such as the matrix. For example, the matrix may be a membrane and the other absorbent material may be a nonwoven or a paper. Other combinations are of course possible as well.

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

The different zones in the sample channel may also differ in that there are zones with capillary activity and those without. In addition, zones of high hydrophilicity and low hydrophilicity may be included. The individual zones can virtually seamlessly pass into each other or through certain barriers, such as valves, in particular non-closing valves, such as geometric valves or hydrophobes be separated from each other.

The reagents in the sample channel are preferably in dried or lyophilized form. However, it is also conceivable, although less preferred, 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 bottom layer into which the fluidic structures are introduced, and a cover layer, which as a rule contains no further structures apart from the inlet openings for liquids and the vent openings. The introduction of reagents during the preparation of the test device is usually carried out before the upper part of the test element (cover layer) is applied to the lower part (bottom layer). At this time, the fluidic structures are open in the lower part, so that a dosage of the reagents in liquid or dried form is readily possible. The introduction of the reagents can be done for example by printing or dispensing. It is also possible to incorporate the reagents into the test element by placing them in the test element impregnated in absorbent materials such as papers, nonwovens or membranes. After placing the reagents and inserting the absorbent materials, such as the porous, absorbent matrix (membrane) and optionally other absorbent materials (non-woven waste etc.) upper and lower part of the test element are connected to each other, for example, clipped, welded, glued and such more.

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

Typically, the sample channel contains a zone for separating particulate matter from the liquid sample. In particular, if blood or other bodily fluids containing cellular components are used as the sample material, this zone serves to separate the cellular sample components. From blood can be so by separating in particular the red blood cells (erythrocytes) almost colorless plasma or serum which is usually better suited for subsequent visual or optical detection methods than the strongly colored blood.

Preferably, the separation of cellular sample components by centrifugation, d. H. by fast rotation of the test element after filling with liquid sample. The test element according to the invention contains suitably dimensioned and geometrically designed channels and / or chambers for this purpose. 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).

To control the flow of the sample liquid in the test element, it may contain valves, in particular so-called non-closing or geometric valves or hydrophobic barriers, especially in the sample channel.

The valves or hydrophobic barriers can be used to 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 may have a sample metering zone, which allows a precise measurement of the - initially in excess abandoned - sample. In a preferred embodiment, the sample metering 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 receive an excess of sample material. The sample, either driven by capillary forces or by centrifugation, flows 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 to the valve, a sample excess chamber adjacent to the sample application zone and branching off from the sample channel is filled, for example by capillary forces or by centrifuging the test element. It must be ensured that the sample volume to be measured is initially not transported across the valve by suitably selecting the valve. As soon as excess sample is trapped in the appropriate transfer chamber, there is a well-defined sample volume between the valve of the sample channel on one side 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 now extends beyond the valve emotional. All fluidic areas that lie after the valve and come into contact with the sample are now filled with a precisely defined sample volume.

The sample channel may also have an inflow for other liquids except the sample liquid. For example, in the sample channel, a second channel open, the z. B. can be filled with a washing or reagent liquid.

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

Preferably, the optics of the measuring device can be used for fluorescence measurement with spatially resolved detection. For two-dimensional, d. H. planar evaluation optics, is typically used to illuminate the detection area of the test element and possibly the excitation of optically detectable markers an LED or a laser. The detection of the optical signal is by CMOS or CCD (typically 640 x 480 pixels). The beam path is direct or folded (eg 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 be done here via a diode array. In this case, the rotational movement of the device can be utilized for the illumination and evaluation of the second dimension in order to scan with the diode array over the area of the test element to be evaluated.

As drive for rotating and positioning of the test element, a DC motor with encoder or a stepper motor can be used.

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

The method according to the invention serves to detect an analyte in a liquid sample. In this case, the sample is first introduced into the sample application opening of the test element according to the invention. Subsequently, the test element is rotated about its central axis: The sample is transported from the sample application opening to the off-axis end of the porous, absorbent matrix. The rotation of the test element then becomes so far slows or stops that the sample or a material obtained from the sample as it flows through the test element from the off-axis to the near-axis end of the porous, absorbent matrix is sucked. The analyte is finally visually or optically detected in the porous, absorbent matrix or a downstream zone.

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

It is also possible that after the rotation of the test element, a further liquid is applied to the test element, which is sucked after the sample from the off-axis to the near-axis end of the porous, absorbent matrix.

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

The invention has the following advantages

The combination of liquid transport by means of centrifugal forces and suction forces in porous, absorbent matrix materials allows precise control of liquid flows. According to the invention, the porous, absorbent matrix transports the liquid from an off-axis end to an off-axis end, i. H. 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 exactly. Targeted control of the rotation of the test element therefore makes it possible to slow down or stop the flow of the sample liquid in the porous, absorbent matrix, so that targeted and defined reaction conditions can be maintained. At the same time, the use of the porous absorbent matrix, which serves essentially as a scavenger matrix for bound-free separation in immunoassays, allows efficient capture of sample components during the immunoassay.

The invention is explained in more detail by the following examples and figures. In each case reference is made to immunological sandwich assays. However, the invention is not limited thereto. It can be applied to other types of immunoassays, in particular competitive immunoassays, or other types of specific binding assays (for example, those which use sugar and lectins as binding partners, hormones and their receptors, or complementary pairs of nucleic acids) are also used. Typical representatives of these specific binding assays are known per se to the person skilled in the art (in connection with immunoassays, express reference is made here to FIGS. 1 and 2 and the associated description passages of the document US 4,861,711 And can be readily applied to the present invention.

FIG. 1 shows a schematic representation of a plan view of a preferred embodiment of the test element according to the invention. For the sake of clarity, only the layer of the test element which contains the fluidic structures is shown. The embodiment shown here contains only one opening for introducing sample and / or washing liquid. The separation of interfering sample components is done in this embodiment) after the sample has been contacted with reagents.

FIG. 2 schematically shows a further preferred embodiment of the test element according to the invention. Again, only the structure has been shown, which has the fluidic elements of the test element. In this embodiment of the test element there are two separate sample and wash buffer application ports. A separation of the cellular sample components takes place here already before the sample is brought into contact with reagents.

FIG. 3 shows a variant of the embodiment according to FIG. 1 in a schematic representation. Again, the separation of cellular sample components, after which the sample was brought into contact with reagents. However, the structure according to FIG. 3 has a separate feed for washing liquid.

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

FIG. 5 represents a slight further development of the test element according to FIG. 3. In contrast to the embodiment according to FIG. 3, FIG. 5 contains a different geometric arrangement of the waste nonwoven and another type of valve at the end of the sample metering section.

FIG. 6 schematically shows a plan view of a further development of the test element according to FIG. 5. In contrast to the embodiment according to FIG. 5, the embodiment according to FIG. 6 contains a fluidic structure for receiving a sample excess.

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

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

FIG. 9 schematically shows a plan view of an alternative to the test element according to FIG. 6. In contrast to the embodiment according to FIG. 6, the embodiment according to FIG. 9 contains an off-axis sample application opening, which initially brings the sample nearer to the center of the test element via a capillary, that is to say in FIG an off-axis area.

FIG. 10 shows the typical curve for troponin T measurements in whole blood samples (concentration of troponin T in ng / ml plotted against the signal strength (counts)). The samples were supplemented with recombinant troponin T to the respective concentration. The data belong to Example 2 and were obtained with the aid of test elements according to FIG. 6 / Example 1.

1

scheibenförmiges Testelement (Disk)

2

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

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

disk-shaped test element (disk)

2

Substrate (eg one or more parts, injection molding, milled, built up from layers ...)

3

central recess (drive hole)

4

Sample application opening

5

Sample dosing zone (dosing section of the channel)

6

Capillary stop (eg hydrophobic barrier, geometric / non-closing valve)

7

Container for sample surplus

8th

Capillary stop (eg 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 (fleece)

14

Capillary stop (eg hydrophobic barrier, geometric / non-closing valve)

15

channel

16

Addition opening for other liquids, eg. B. wash buffer

17

vent

18

Decanting channel

19

Capillary stop (eg hydrophobic barrier, geometric / non-closing valve)

20

collection reservoir

21

capillary

FIGS. 1 to 9 show different preferred embodiments of the test element (1) according to the invention. In essence, each of the substrate (2) containing the fluidic structures and the central recess (drive hole 3) is shown. In addition to the substrate, which can be one or more parts, for example, and can be constructed by means of injection molding, milling or by laminating corresponding layers, the disk-shaped test element (1) according to the invention also generally comprises a cover layer, which in the figures provides clarity is not shown half. In principle, the cover layer can also carry structures, but as a rule it will have no structures other than the openings for the samples and / or further liquids to be dispensed onto the test element. The cover layer can also be designed completely without openings, for example in the form of a film, which is connected to the substrate and terminates the structures located therein.

The embodiments illustrated in FIGS. 1 to 9 show fluidic structures that perform largely similar functions, although they differ in detail from embodiment to embodiment. The basic structure and the basic function will therefore be explained in more detail here with reference to the embodiment according to FIG. The embodiments according to FIGS. 2 to 9 will subsequently be explained in more detail merely with reference to the specific differences in order to avoid unnecessary repetitions.

FIG. 1 shows a first preferred embodiment of the disk-shaped test element (1) according to the invention. The test element (1) contains a substrate (2) containing the fluidic and microfluidic and chromatographic structures. The substrate (2) is covered by a corresponding counterpart (cover layer) (not shown), which contains sample application and ventilation openings which lead to the structures in the substrate (2). correspond. 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, makes it possible to rotate the disk-shaped test element (1). Alternatively, it is possible that the test element (according to one of FIGS. 1 to 9) has no such central recess (3) and the drive is rotated via a drive unit of the measuring device designed in accordance with the outer contours of the test element, for example a rotating plate into which the test element is inserted into a recess corresponding to its shape.

Sample liquid, in particular whole blood, is supplied 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 metering zone (5) can also contain the dried reagents. It is limited by the Kapillarstopps (6 and 8), which may be formed for example as a hydrophobic barrier or as a geometric / non-closing valve. The limitation of the sample dosing zone (5) by the capillary stops (6, 8) ensures that a defined sample volume is taken up and passed on into the fluidic zones which lie downstream of the sample dosing zone (5). Upon rotation of the test element (1), any sample excess is transferred from the sample application port (4) and sample dosing zone (5) to the sample overflow container (7) while the measured amount of sample from the sample dosing zone (5) enters the channel (9 ) is transferred.

At appropriate rotational speeds, channel (9) is used to start the separation of red blood cells and other cellular sample components. The reagents contained in the sample dosing zone (5) are already dissolved in the sample when the sample enters the channel (9). The entry of the sample in channel (9) via the capillary stop (8) leads to a mixing of the reagents in the sample.

The possibility of temporal control of the rotation processes that are possible with the test element according to the invention, thereby allowing a targeted control of the residence times and thus the incubation time of the sample with reagents and the reaction times.

During 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 process Erythrocyte collection zone (11) while the plasma remains substantially in the collection zone (10).

In contrast to test elements, the membranes or nonwovens for separating particulate sample components use (for example, glass fiber webs 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 exploited much more effectively with the test elements according to the invention, as there are virtually no dead volumes (eg, volume of fiber interstices or pores) from which the sample can not be withdrawn. In addition, these prior art blood separation membranes and webs tend in part to undesirably adsorb sample components (e.g., proteins) or destroy (lysate) cells, which is also not observed with the test elements of the present invention.

If the rotation of the test element (1) is stopped or slowed down, the reagent-plasma mixture (which has formed in the presence of the analyte in the case of an immunoassay, for example sandwich complexes of analyte and antibody conjugates) by the suction of the porous-absorbent matrix (12) incorporated into and passed through them. In the case of immunoassays, the immobilized binding partners contained in the membrane (12) capture the analyte-containing complexes in the detection zone and bound unbound labeled conjugate in the control zone. The nonwoven (13) adjoining the porous, absorbent matrix supports the movement of the sample through the membrane (12). The fleece (13) also serves to receive the sample after flowing 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), in a subsequent step, wash buffer is pipetted into the sample application opening (4). By means of the same combination of capillary, centrifugal and chromatographic forces, the washing buffer flows through the corresponding fluidic structures of the test element (1) and, in particular, washes the membrane (12), where the bound analyte complexes are now located, thus removing excess reagent residues. The washing step may be repeated one or more times so as to improve the signal-to-background ratio. This allows an optimization of the detection limit for the analyte and an increase of the dynamic measuring range.

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 comprises In this case, 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 embodiments, the sample channel may consist of more or fewer individual zones / chambers.

FIGS. 3, 5, 6, 7 and 9 show essentially analogous embodiments to FIG. 1. FIG. 3 differs from FIG. 1 in that on the one hand, no container for excess sample (7) adjoins the sample application opening (4) and at the end of the Probenendosierabschnitts (5) no capillary stop is present (ie here a metered sample application is required) and on the other hand, that a separate addition port (16) for other liquids, such as. As 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 FIG. 5 is largely identical to the embodiment according to FIG. 3. The two embodiments differ only in the shape of the waste fleece (13) and in that the test element according to FIG. 5 has a capillary stop (8) at the end of the sample-metering section (FIG. 5).

The embodiment according to FIG. 6 is again essentially identical to the embodiment according to FIG. 5 and differs therefrom by the additional presence of a container for sample excess (7) in the region between the sample metering opening (4) and the sample metering zone (5). Here, no metered application of the sample is required (analogous to FIG. 1).

The embodiment of the test element (1) according to the invention according to FIG. 7 substantially corresponds to the test element (1) of FIG. 6. Both embodiments have the same fluidic structures and functions. Only the arrangement and geometric design is different. The embodiment according to FIG. 7 has additional ventilation openings (17), which are necessary due to the different dimensioning of the fluidic structures in comparison to FIG. 6 in order to allow filling of the structures with samples or washing liquid. Channel (9) is designed here as a thin capillary, which is filled only during rotation of the test element (ie overcoming the capillary stop (8) is possible only by means of centrifugal force). Already during the rotation, it is possible with the test element (1) according to FIG. 7 to remove recovered plasma from the erythrocyte collection zone 11; This is done by the decanter unit 18, which finally discharges into the serum / plasma collection zone 10.

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

In contrast to the embodiment according to FIGS. 1, 3, 5, 6, 7 and 9, in the embodiments according to FIGS. 2, 4 and 8 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 a sample material does not lead to different measurement results, since always initially plasma or serum comes into contact with the reagents and the dissolution / incubation / reaction behavior should be practically the same , Also in the embodiments according to FIGS. 2, 4 and 8, the liquid sample is first applied to the test element (1) via the sample application opening (4). By capillary forces and / or centrifugal forces, the sample is then transported from the sample application opening (4) in the channel structures. In the embodiments according to FIGS. 2 and 4, the sample is transferred into a sample metering section (5) after it has been introduced into the sample application opening (4), and then a serum / plasma separation from the whole blood is effected by rotation. The unwanted cellular sample constituents, essentially erythrocytes, accumulate in the erythrocyte trap (11), while serum or plasma accumulate in the 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 accommodated and are dissolved when the sample flows in. By rotating the test element (1) again, the sample-reagent mixture of channel structure (9) can overcome the capillary stop (14) and thus reach the membrane (12) via the channel (15). When slowing or stopping the rotation, the sample-reagent mixture is transported via the membrane (12) into the waste nonwoven (13).

The embodiments according to FIG. 2 and FIG. 4 differ from one another in that a container for sample excess (7) is provided in FIG. 2, while the embodiment according to Figure 4 does not provide such a function. As in the embodiment according to FIG. 3, a metered application of the sample is expedient here.

FIG. 8 shows a variant of the embodiments according to FIGS. 2 and 4. Here, immediately after the sample application opening (4), after passing through a first geometric valve (19), the sample is transferred by centrifugation into an erythrocyte separation structure (10, 11). The area denoted by (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 centrifuging. Chamber (20) serves as a collecting reservoir for excess serum or plasma, which after complete filling of the sample dosing section (5) possibly flows from the serum / plasma collection zone (10). All other functions and structures are analogous to FIGS. 1 to 7.

By deliberately 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 are moved either only with the aid 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 already described above in connection with FIG. 1, the test elements according to the invention according to FIGS. 1, 2, 6, 7, 8 and 9 have an automatic functionality which allows a relatively accurate measurement of a sample aliquot from a sample which has been applied in excess to the test element allowed (so-called "metering system"). This metering system is a further subject of the present invention. It essentially comprises the elements 4, 5, 6, and 7 of the illustrated test elements (1). Sample liquid, in particular whole blood, is supplied 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 metering zone (5) can also contain the dried reagents. It is limited by the Kapillarstopps (6 and 8), which may be formed for example as a hydrophobic barrier or as geometric / non-closing valves. The limitation of the sample dosing zone (5) by the capillary stops (6, 8) ensures that a defined sample volume is taken up and passed on into the fluidic zones which lie downstream of the sample dosing zone (5). Upon rotation of the test element (1), a possible excess sample from the sample application opening (4) and the sample dosing (5) is transferred to the sample surplus container (7) while the measured amount of sample is transferred from the sample dosing zone (5) to the channel (9). Alternatively, it is also possible to use other forces instead of the force generated by rotation, which moves the sample, for. B. by applying an overpressure on the sample input side or a negative pressure on the sample output side. Consequently, the illustrated metering system is not necessarily bound to rotatable test elements, but can also be used in other test elements.

For example, similar metering systems are off US 5,061,381 known. Also in this document, a system is described in which sample liquid can be applied in excess to a test element. The measurement of a relatively accurate sample aliquot, which is then further processed in the test element, also takes place here by the interaction of a metering chamber and an overflow chamber, these two zones - unlike the present invention - by a narrow, but at least during filling always liquid exchange enabling contact. Sample liquid is here directly separated during filling of the test element in a part which is passed through a wide channel in the "metering chamber", and a part which flows through a narrow channel in the "overflow chamber". After complete filling of the "metering chamber", the test element is set in rotation and any excess sample is diverted into the "overflow chamber" so that only the desired, measured sample volume remains in the "metering chamber", which is subsequently processed further.

A disadvantage of the embodiment of the metering system according to US 5,061,381 is that for sample volumes that are placed on the test element and which correspond exactly to the minimum volume or only slightly larger than the minimum volume, there is a risk that the dosing is underdosed, since always a portion of the sample from the beginning unhindered in the "overflow chamber" flows.

This problem is solved in the presently proposed embodiment of the metering system in that a capillary stop (hydrophobic barrier or a geometric or non-closing valve) is arranged between the metering zone and the zone for the sample excess. When filling the test element with sample, therefore, the sample is first conducted almost exclusively in the dosing. The capillary stop prevents sample from entering the sample excess zone before the sample dosing zone is completely filled. Also for 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, it is ensured that the sample dosing zone is completely filled.

example 1

Production of a test element according to FIG. 6

1.1. Production of the substrate (2)

By means of injection molding, a substrate (2) according to FIG. 6 is produced from polycarbonate (PC) (alternatively polystyrene (PS), ABS plastic or polymethyl methacrylate (PMMA) is also possible as material) (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 figures refer to FIG. 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 = 26.5 mm ³ No.8: t = 500 μm No.9: t = 110 μm No. 10: t = 550 μm No. 11: t = 130 μm; V = 15 mm ³ No. 15: t = 150 μm; V = 11.4 mm ³

A transition from shallower to deeper structures is usually only possible for liquids in the fluidic structures, if external force (eg centrifugal force) acts on them. 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 addition 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 by means of plasma treatment and hydrophilized.

1.2. Introduction of the reagents

Part of the reagents required for analyte detection (eg biotinylated anti-analyte antibodies and anti-analyte antibodies labeled with a fluorescence label) are introduced into the sample dosing section (5) alternately as a solution by means of piezo-dosing as a solution and then dried, so that virtually its entire inner surface is occupied with reagents.

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

1.3. Inserting the membrane (12)

In a corresponding recess in the substrate (2) is the porous matrix (12) (nitrocellulose membrane on plastic carrier film, 21x5 mm ² , reinforced with 100 micron PE film cellulose nitrate membrane (type CN 140 Sartorius, Germany)), in which by means of line impregnation (see below) an analyte detection line (polystreptavidin) and a control line (polyhapten) was introduced, inserted and optionally fixed by means of double-sided adhesive tape.

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

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

1.4. Apply the cover

Subsequently, the cover (film or injection molded part without Fluidikstrukturen, which may or may be hydrophilized) is applied and optionally permanently connected to the substrate (2), preferably glued, welded or clipped.

1.5. Loading the Waste Fleece (13)

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

By the quasi self-dosing sample receiving unit (comprising the sample application opening (4), the sample dosing (5) and the structures limiting it (capillary stop (8) and container for excess sample (7)) ensures that regardless of the on the test element (1) discontinued Amount of sample (if it exceeds a minimum volume (in this example 27 μl)) if different test elements are used, reproducibly the same amount of samples.

Through the distribution of the reagents in the entire sample metering section (5), preferably in the form of alternating reagent spots (ie smaller, almost punctiform reagent areas), in combination with a rapid filling of the sample metering 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, a virtually complete dissolution of the reagents, so that here again an increased reproducibility compared to conventional, based on absorbent materials test elements (test strips, bio-discs with reagent pads, etc.) is observed.

Example 2 Detection of troponin T with the aid of the test element from Example 1

On the test element according to Example 1, 27 .mu.l of whole blood to which different amounts of recombinant troponin T were added, abandoned. The test element is then further treated on the basis of the procedure given in Table 1 and finally measured the fluorescence signals for different concentrations. Table 1: Measurement procedure Time (min: sec) Duration (min: sec) Rotation at revolutions per minute action 00:00 01:00 0 Add 27 μl sample; Dissolve the reagents 01:00 02:00 5000 Erythrocyte separation and incubation 03:00 01:00 800 Chromatography (generate signal) 04:00 12:10 0 Add 12 μl washing buffer ¹ ) 04:10 02:00 800 Wash buffer transport and chromatography 06:10 12:10 0 Add 12 μl washing buffer ¹ ) 06:20 02:00 800 Wash buffer transport and chromatography 08:20 12:10 0 Add 12 μl washing buffer ¹ ) 08:30 02:00 800 Wash buffer transport and chromatography 10:30 0 measure up ¹⁾ 100 mM Hepes, pH 8.0; 150 mM NaCl; 0.095% sodium azide.

The measured data are shown in FIG. 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 reference method "Roche Diagnostics Elecsys Troponin T Test".

Compared to conventional immunochromatographic troponin T-test strips, such as. 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 downwards (Cardiac Troponin T: 0.1 ng / ml, invention: 0.02 ng / ml) and the dynamic measuring range after dilated at the top (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 (19)

Test element, which is substantially disc-shaped and flat and is rotatable about a central axis which is perpendicular to the disc-shaped test element plane, containing a sample introduction opening for dispensing a liquid sample, a porous, absorbent matrix with a first end remote from the axis, and a sample channel which extends from the sample introduction opening over an area near the axis to the off-axis first end of the porous, absorbent matrix, characterized in that
the porous, absorbent matrix further comprises an axially proximal second end. Test element according to claim 1, characterized in that
the sample application opening is near the axis and the sample channel extends from the near-axis sample application opening to the off-axis first end of the porous, absorbent matrix. Test element according to claim 1, characterized in that
the sample application opening is remote from the axis and is connected via a capillary channel to an area near the axis. Test element according to claim one of the preceding claims, characterized in that
the porous, absorbent matrix is a paper, a membrane or a non-woven. Test element according to claim one of the preceding claims, characterized in that
the porous, bibulous matrix contains one or more zones of immobilized reagents. Test element according to one of the preceding claims, characterized in that the porous, absorbent matrix is in contact with the second end close to the axis with another absorbent material which can receive liquid from the matrix. Test element according to one of the preceding claims, characterized in that the sample channel contains zones of different dimensions and / or for different functions. Test element according to one of the preceding claims, characterized in that the sample channel contains a zone containing soluble reagents. Test element according to one of the preceding claims, characterized in that the sample channel contains a zone for separating particulate components from the liquid sample. Test element according to one of the preceding claims, characterized in that the sample channel contains geometric valves or hydrophobic barriers. Test element according to one of the preceding claims, characterized in that the sample channel contains a sample metering zone. Test element according to one of the preceding claims, characterized in that the sample channel has an inflow for other liquids except the sample liquid. A test element according to any one of claims 1 to 12, characterized in that the sample introduction port is in contact with a sample metering zone and a sample overflow zone with capillary stop between the sample metering zone and the sample overflow zone. A test element comprising a sample application port, a sample metering zone and a sample overflow zone, the sample application port being in contact with the sample metering zone and the sample overflow zone, characterized in that there is capillary stop between the sample metering zone and the sample overflow zone. Method for detecting an analyte in a liquid sample, wherein the sample is introduced into the sample application opening of the test element according to one of claims 1 to 13, the test element is rotated about the central axis, so that the sample is transported to the off-axis end of the porous, absorbent matrix, the rotation of the test element is slowed down or stopped so that the sample or a material obtained from the sample as it flows through the test element is sucked from the off-axis to the near-axis end of the porous, absorbent matrix, and - The analyte is detected visually or optically in the porous, absorbent matrix or a downstream zone. A method according to claim 15, characterized in that
after the rotation of the test element, a further liquid is applied to the test element, which is sucked after the sample from the off-axis to the near-axis end of the porous, absorbent matrix. A method according to claim 15 or 16, characterized in that the migration of the liquid sample and / or the further liquid over the matrix is selectively slowed down or stopped by the rotation of the test element A system for determining an analyte in a liquid sample comprising a test element according to any one of claims 1 to 13 and a measuring device, wherein the measuring device - At least one drive for the rotation of the test element and - Has an evaluation optics for evaluating the visual or optical signal of the test element. Use of a test element according to one of claims 1 to 14 or a measuring system according to claim 18 for the determination of an analyte in a liquid sample.

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